Journal articles: 'Alternative last exon (ALE)'

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Lee, Seungjae, Lu Wei, Binglong Zhang, Raeann Goering, Sonali Majumdar, Jiayu Wen, J. Matthew Taliaferro, and Eric C. Lai. "ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing." PLOS Genetics 17, no. 4 (April 7, 2021): e1009439. http://dx.doi.org/10.1371/journal.pgen.1009439.

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ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member,DrosophilaElav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets ofDrosophilaare known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants ofelav/fne, but notelavalone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation ofDrosophilaELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3’ UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification inDrosophilaand mammalian neurons.

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Mohamed, Aminetou Mint, Morgan Thenoz, Catherine Koering, Pierre Mallinjoud, Didier Auboeuf, Francoise Solly, Meyling Cheok, et al. "DEK and WT1 Affect Alternative Splicing of Genes Involved in Hematopoietic Cell Lineage and Resistance to Chemotherapy in Acute Myeloid Leukemia Cells." Blood 120, no. 21 (November 16, 2012): 2392. http://dx.doi.org/10.1182/blood.v120.21.2392.2392.

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Abstract Abstract 2392 In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in acute myeloid leukemia (AML). In MDS the frequency of somatic spliceosomal mutations (SSM) range from 1–3% for U2AF1 in RARS/RCMD-RS to more than 70% for SF3B1 in ARSI. These values are significantly lower in AML whereas AML cells cumulate numerous splicing defects. Beside SSMs, one can propose that alternative splicing (AS) might be disturbed by other processes such as abnormal protein-protein interactions. DEK and WT1 are 2 oncogenes overexpressed in most patients with AML. They physiologically influence AS through physical interactions with the heterodimer U2AF1/U2AF2 involved in the recognition of splice acceptor site by the splicing machinery. It is therefore possible that the leukemogenic overexpression of DEK or WT1 might deregulate AS in AML cells, even in the absence of SSM. Here we show that DEK and WT1 affect AS in AML cells. Exon expression profiling was performed in triplicate with MOLM13, KASUMI and KG1 AML cells stably knocked down or not for DEK and WT1 through shRNA. The efficiency of shRNA-mediated silencing was confirmed by western blot and total RNA was analyzed using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). Microarray data were cross-compared between cell lines and only statistically significant modifications (p<0.05) shared by MOLM13, KASUMI and KG1 cells were selected. DEK and WT1 knock-down induced the transcriptional deregulation of 1613 (813 up) and 3280 (998 up) genes in AML cell lines, respectively. AS events were selected and annotated with fasterDB database (http://fasterdb.com/faster/home.pl) for genes displaying either no or low (<2) differential transcription. With this approach, differential expression of DEK coincided with changes in 1049 AS events over 934 genes. Those were distributed in 4 alternative acceptor sites (ACC), 222 first exons (AFE), 257 last exons (ALE), 539 spliced exon (ASE), 6 deletions (DEL), 21 donor sites (DON). Differential expression of WT1 led to modifying 1371 alternative splicing entities over 1198 genes. Those were distributed in 6 ACC, 385 AFE, 343 ALE, 590 ASE, 13 DEL, and 34 DON. Genes with AS events were then sorted based on gene function with DAVID bioinformatics resources version 6.7 (http://david.abcc.ncifcrf.gov/). The results indicated that, in both DEK- and WT1-dependent assays, a large subset of genes were related to hematopoietic cell lineage followed by other functional categories such as calcium signaling, ATP-binding cassette (ABC) transporters, and focal adhesion pathways that have been previously reported to be affected in AML cells and involved in resistance to chemotherapy. Differential expression of WT1 modified AS of CD3E, CD9, CSF1R, CSF3R, CR2, GP1BA, ITGA1, ITGA3, ITGB3, IL1A and IL6 while that of DEK led to modulate AS of CD19, CD1d, CD36, CD3G, CSF1R, CR2, ITGA1, ITGA2B, ITGA4, ITGA6, IL7, HLA-DRB5, and MME. Microarray data were validated by exon specific RT-PCR. Exon expression profiling of fresh AML bone marrow samples with or without U2AFs mutations and various levels of DEK or WT1 expression is currently in progress and will be presented. In conclusion in AML cells, DEK and WT1 overexpression affects AS of numerous key genes involved in hematologic differentiation, leukemogenesis and resistance to chemotherapy. These posttranscriptional effects occur in the absence of transcriptional change and therefore highlight hitherto unknown phenotypic alterations having putative diagnostic, prognostic and therapeutic interests. DEK and WT1 are overexpressed in more than 80% of AML and target AS via U2AFs that are mutated in only 0–8% of cases. Accordingly present results strongly suggest that other factors than SSM divert AS in AML. Disclosures: No relevant conflicts of interest to declare.

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Levallet, J., H. Mittre, B. Delarue, and S. Carreau. "Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells." Journal of Molecular Endocrinology 20, no. 3 (June 1, 1998): 305–12. http://dx.doi.org/10.1677/jme.0.0200305.

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Expression of cytochrome P450 mRNA in rat germ cells was characterized by reverse transcription PCR with various primers located at the 3'-end of the coding region. At least two unusual isoforms (Ex10-S and INT) of P450 aromatase (P450arom) mRNA were expressed. Analysis of their sequences demonstrated that an alternative splicing event occurred first at the exon-intron boundary of the GT consensus sequence of the last coding exon, and second in the internal 5' donor inside exon 9 used as a minor cryptic splicing site. These isoforms lacked the last coding exon which contained the heme-binding domain; in addition, for the Ex10-S transcript, the catalytic domain was also absent because of a frameshift in the open reading frame. The deduced amino acid sequences led to truncated P450arom polypeptides without the heme-binding domain, which were probably unable to convert androgens into estrogens. Adult rat germ cells are able to express P450arom mRNA, which is then translated into a biologically active enzyme which is involved in estrogen production. Moreover, for the first time, we report the existence of alternative splicing events of P45Oarom mRNA in pachytene spermatocytes and round spermatids, which probably cannot encode functional aromatase molecules.

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Hu, Zhigang, Junting Cao, Liyan Ge, Jianqin Zhang, Huilin Zhang, and Xiaolin Liu. "Characterization and Comparative Transcriptomic Analysis of Skeletal Muscle in Pekin Duck at Different Growth Stages Using RNA-Seq." Animals 11, no. 3 (March 16, 2021): 834. http://dx.doi.org/10.3390/ani11030834.

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Skeletal muscle, accounting for approximately 50% of body weight, is the largest and most important tissue. In this study, the gene expression profiles and pathways in skeletal muscle of Pekin duck were investigated and compared at embryonic day 17, 21, and 27 and postnatally at 6 months of age. An average of 49,555,936 reads in each sample was obtained from the transcriptome libraries. Over 70.0% of alternative splicing (AS) in each sample was mainly alternative 5′ first exon (transcription start site)—the first exon splicing (TSS) and alternative 3′ last exon (transcription terminal site)—the last exon splicing (TTS), indicating that TSS and TTS were the most common AS event in Pekin ducks, and these AS events were closely related to the regulation of muscle development at different growth stages. The results provided a valuable genomic resource for selective breeding and functional studies of genes. A total of 299 novel genes with ≥2 exons were obtained. There were 294 to 2806 differentially expressed genes (DEGs) in each pairwise comparison of Pekin duck. Notably, 90 DEGs in breast muscle and 9 DEGs in leg muscle were co-expressed at all developmental points. DEGs were validated by qPCR analysis, which confirmed the tendency of the expression. DEGs related to muscle development were involved in biological processes such as “endodermal cell differentiation”, “muscle cell cellular homeostasis”, “skeletal muscle tissue growth” and “skeletal muscle cell differentiation”, and were involved in pathways such as oxidative phosphorylation, ECM-receptor (extracellular matrix receptor) interaction, focal adhesion, carbon metabolism, and biosynthesis of amino acids. Some DEGs, including MYL4, IGF2BP1, CSRP3, SPP1 and KLHL31, as well as LAMB2, LAMA2, ITGB1 and OPN, played crucial roles in muscle growth and development. This study provides valuable information about the expression profile of mRNAs and pathways from duck skeletal muscle at different growth stages, and further functional study of these mRNAs and pathways could provide new ideas for studying the molecular networks of growth and development in duck skeletal muscle.

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Vreken, Peter, René W. L. M. Niessen, Marjolein Peters, Marianne C. L. Schaap, Johanna G. M. Zuithoff-Rijntjes, and Augueste Sturk. "A Point Mutation in an Invariant Splice Acceptor Site Results in a Decreased mRNA Level in a Patient with Severe Coagulation Factor XIII Subunit A Deficiency." Thrombosis and Haemostasis 74, no. 02 (1995): 584–89. http://dx.doi.org/10.1055/s-0038-1649779.

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SummaryAmplification and sequencing of exons I-XV of the gene encoding subunit A of coagulation factor XIII (FXIII) in a patient with severe subunit A deficiency revealed a single G → A base substitution at the last position of intron E, mutating the invariant AG dinucleotide splice acceptor site to AA. Northern blot analysis of FXIII subunit A mRNA levels in peripheral mononuclear leukocytes showed that this mutation leads to an undetectable FXIII subunit A mRNA level, suggesting that the mutant transcript is either highly unstable or only spliced at low efficiency. Despite this low mRNA level we were able to amplify cDNA fragments containing the exonV-exonVI junction. Sequence analysis showed that the AA dinucleotide is not recognized by the splicing machinery. Instead, an AG dinucleotide located seven bases downstream of the mutated splice acceptor site is used as alternative acceptor. The resulting, alternatively spliced, FXIII subunit A transcript contains a deletion of the first seven bases of exon VI, while translation continues out of frame and leads to a premature stop codon 27 bases thereafter.

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Bernasconi, Paolo, Barbara Rocca, Celeste Calvello, Catherine Klersy, Marina Boni, Rita Zappatore, Irene Dambruoso, et al. "Alternative Splicing of hTERT Exon 7 in AML: Biological Fuction and Prognostic Significance." Blood 124, no. 21 (December 6, 2014): 1019. http://dx.doi.org/10.1182/blood.v124.21.1019.1019.

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Abstract hTERT, a telomere specific reverse transcriptase with a crucial role in telomerase activity, prevents telomeres shortening, a pro-apototic cellular event which occurs at every round of DNA replication. Its regulation is complex as alternative spliced variants which generate the full-length hTERT transcript (+α+β) and transcripts carrying α and/or β deletions (transdominant negative isoform -α+β, inactive products +α-β and -α-β) have been reported. In AML the prognostic significance of hTERT expression is still debated, even if its over-expression has been correlated with a poor clinical outcome. In contrast, up to now no study has dealt with the frequency and the prognostic significance of variant transcripts. Thus, our study was aimed at estimating hTERT isoforms distribution in a series of de novo AML patients (pts), at establishing whether their expression varied between pts with a normal and a complex chromosome pattern, at estimating their prognostic significance. The ninety-seven de novo AML pts included in the present study came to our observation in the period January 2010 and January 2013. They were thirty-eight females and fifty-nine males; their median age was 59 years (range 18-84). On conventional cytogenetic studies fifty-two, whose median age was 56 years (range 24-84), presented a normal karyotype and forty-five, whose median age was 60 years (range 33-84), presented a complex karyotype (≥3 defects); the two pts groups were comparable by age. According to WHO classification 6 (6.1%) pts were classified as M0/M1, 47 (48.4%) as M2, 38 (39.1%) as M4, 5 (5.1%) as M5 and one as M6. An internal tandem duplication (ITD) of the FLT3 gene and a NPM1 mutation were revealed in 12 and 2 chromosomally normal pts. No CK presented a FLT3 ITD. All pts received standard induction chemotherapy followed by two courses of consolidation treatment. At the time of the study 38 pts achieved a complete remission (CR) and 36 died. Median follow-up was 22.7 months (range: 13.5-60.2). hTERT isoforms expression was determined in bone marrow samples by real-time reverse transcriptase polymerase chain reaction, using SYBR Green I. hTERT transcript (+α+β) primers’ design was made using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), while for the other primers we referred to Capraro et al. 2011. Specific amplifications were confirmed by sequences analysis. In order, to estimate hTERT isoforms expression levels in normal mononuclear cells, twenty-three umbilical cord bloods (UCB) were examined (control group). When the Kruskal-Wallis rank test was applied to compare the expression of hTERT isoforms inAML pts with a normal and a complex karyotype no significant difference was observed (P=0.39, P=0.77; +α+β, -α+β respectively). Instead, independently from karyotype, in Cox univariate analysis, pts who over-expressed the trans-dominant negative isoform (-α+β) experienced a significant better response to treatment than the other pts (P=0.03, HR=2.11; 95% CI:1,13-3,93). In addition, pts who over-expressed inactive products (-α-β) (first group) presented a leukemia-free survival inferior to that of pts who presented a low expression of these products (second group) (P=0,003, HR=3,26; 95% CI:1,33-7,96). Moreover, pts who over-expressed the full-length hTERT transcript (+α+β) and the trans-dominant negative isoform (-α+β) presented only a trend towards significance. The first group of pts presented a worse overall survival (HR=1,66; 95% CI:0.94-2,91), whereas the second group a better overall survival (HR=0,58; 95% CI:0,33-1,02). We conclude that the over-expression of the hTERT trans-dominant negative isoform identifies pts with low-risk AML, whereas the over-expression of the hTERT inactive product (-α-β) identifies pts with high-risk AML. In addition, this last hTERT isoform might control the expression of its trans-dominant negative isoform. Thus, our results suggest an intriguing link between the control of hTERT isoforms expression and AML outcome. Disclosures No relevant conflicts of interest to declare.

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Arnaud, Pauline, Margaux Cadenet, Zakaria Mougin, Carine Le Goff, Sébastien Perbet, Mathilde Francois, Sophie Dupuis-Girod, Catherine Boileau, and Nadine Hanna. "Early-Onset Aortic Dissection: Characterization of a New Pathogenic Splicing Variation in the MYH11 Gene with Several In-Frame Abnormal Transcripts." Human Mutation 2023 (August 14, 2023): 1–7. http://dx.doi.org/10.1155/2023/1410230.

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Rare pathogenic variants in the MYH11 gene are responsible for thoracic aortic aneurysms and dissections. They are usually heterozygous missense variants or in-frame deletions of several amino acids without alteration of the reading frame and mainly affect the coiled-coil domain of the protein. Variants leading to a premature stop codon have been described in patients with another phenotype, megacystis-microcolon-intestinal hypoperistalsis syndrome, with an autosomal recessive inheritance. The physiopathological mechanisms arising from the different genetic alterations affecting the MYH11 gene are still poorly understood. Consequently, variants of unknown significance are relatively frequent in this gene. We have identified a variant affecting the consensus donor splice site of exon 29 in the MYH11 gene in a patient who suddenly died from an aortic type A dissection at the age of 23 years old. A transcript analysis on cultured fibroblasts has highlighted several abnormal transcripts including two in-frame transcripts. The first one is a deletion of the last 78 nucleotides of exon 29, corresponding to the use of a cryptic alternative donor splice site; the second one corresponds to an exon 29 skipping. Familial screening has revealed that this molecular event occurred de novo in the proband. Taken together, these experiments allowed us to classify this variant as pathogenic. This case underlines the challenging aspect of the discovery of variations in the MYH11 gene for which the consequences on splicing should be systematically studied in detail.

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Duarte, Adriana S. S., Manoela M. Ortega, Fernando F. Costa, Carmen S. P. Lima, and Sara T. O. Saad. "PP2500 mRNA, a Splice Variant of the Multiple Ankirin Repeat Single KH Domain (Mask), Is Highly Expressed in Plasma Cells of Multiple Myeloma." Blood 106, no. 11 (November 16, 2005): 5090. http://dx.doi.org/10.1182/blood.v106.11.5090.5090.

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Abstract The Ankyrin (ANK)-repeat is one of the most common protein sequence motifs, which leads itself to variation in overall domain size by simple sequence duplication or deletion. The Mask (Multiple Ankyrin Repeats Single KH domain) gene, which codifies an ANK-repeat protein, is located in chromosome 5(q31.3) and it is composed of 39 exons. It generates isoforms by alternative 3′splicing. The first splice variant (hMask) lacks the 10A exon of the Mask gene, generating a mRNA containing 34 exons. The other, Mask-BP3ARF, results from fusion of splice variant hMask, with the two last exons of the gene Eif4Ebp3 (exons B and C) and an intermediate exon (exon 0), generating 36 exons. Recently, a new splice variant, denominated PP2500, was deposited in the data base GeneBank, it presents the first 10 exons, homologous to Mask mRNA with a poly(A+) signal and it is a new splice variant of Mask. In Drosophila, MASK protein seems to interact with members of the Receptor Tyrosine Kinase (RTK) signalling pathway and loss of this interaction increases programmed cell death, reduces cell proliferation, inhibits photoreceptor differentiation, affects RTK dependents processes but does not affect MAPK (Mitogen Activated Protein Kinase) activation. However, the biological functions of these proteins in humans remain still unknown. The aim of this study was to investigate the expression of Mask splice variants in multiple myeloma (MM). Fifteen patients with MM and 3 normal donors participated in this study. Total RNA was extracted from positively selected plasma cells in magnetic column, by Macs Microbeads antibody anti-CD138 and the percentage of purity of plasma cells varied from 78.38% to 96.02% (average 87.95%). We used as control, total RNA from positively selected plasma cells, from a culture of B normal lymphocytes of bone marrow donors (purity 88.69%). The complementary DNA (cDNA) was analyzed by Real-time detection of amplification, performed in an ABI 5700 Sequence Detector System using SybrGreen PCR Master Mix (qPCR). The b-actin gene was used as endogenous control of the reaction. The mean expression of the mRNA of the hMask and Mask-BP3ARF genes were 3 and 4 times increased, respectively, compared with control. The mean expression of the PP2500 mRNA was 14 times increased, compared with control. Quantification of hMask, Mask-BPARF and PP2500 mRNA was not influenced by age, gender, ethnic origin, stage of the disease, β2-microglobulin, serum creatinine and lactate dehydrogenase values (Fisher’s exact test, P> 0.05). Previously we demonstrated that MASK is associated with SHP2, a protein tyrosine-phosphatase. In MM, SHP2 mediates the anti-apoptotic effect of Interleukin-6 (Chauhan et al. JBC275: 27845, 2000). Interleukin-6 triggers proliferation of MM cells via the MAPK cascade, which includes SHP2 activation. Thus, the increased expression of Mask splice variants in plasma cells of MM suggests that their proteins may be involved in this signaling pathway and provide an insight for novel treatment approaches in MM. Supported by FAPESP and CNPq

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Falkenhorst, Johanna, Rainer Hamacher, Peter Reichardt, Philipp Ivanyi, Bernd Kasper, Peter Hohenberger, Barbara Hermes, et al. "Lower-dosing ponatinib in pre-treated GIST: Results of the POETIG phase II trial." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 11536. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.11536.

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11536 Background: Despite long-lasting responses to imatinib most metastatic gastrointestinal stromal tumors (GIST) eventually progress and subsequent treatments are associated with limited duration of disease control. Ponatinib is a potent KIT inhibitor with a strong activity against secondary mutations in exon 17, including the highly resistant D816 mutations of KIT. Based on the dose-depending toxicity profile we sought to evaluate the safety and activity of lower dosing (30mg) ponatinib in pretreated patients with KIT-mutant GIST. We here report safety data for the whole cohort and first efficacy data for the last line cohort within a planned interim analysis. Methods: This multicenter phase 2 trial (NCT03171389) recruited patients with advanced, unresectable GIST progressing after imatinib (Cohort A/B: absence/presence of KIT Exon 13/14 mutations by plasma sequencing) or imatinib, sunitinib and regorafenib (Cohort C). Patients were treated with 30mg oral ponatinib daily in 4-week-cycles. The primary endpoint was the clinical benefit rate at 16 weeks as measured by mRECIST1.1 criteria. Results: At the cutoff date of 31st Jan 2020, 39 patients were evaluable for safety analysis (25 male, 14 female, median age 60 (38-86) years). Median duration of treatment was 65 (14-699) days. 66.7% of all patients observed Grade 3/4 adverse events (AEs), most common were pain (10/39), hypertension (6/39), GGT or lipase increase (both 5/39), and fever (3/39). One AE of special interest was observed (myocardial infarction, rated not related to study drug) and 20/39 patients experienced at least 1 severe AE (6/39 possibly related to ponatinib). Within the last line cohort, 20 patients were evaluable for efficacy. Clinical benefit rate was 35% (CI 15.4-59.2%). Median progression-free survival was 86 days with single patients yielding long-lasting responses (75% quartile 210 days, maximum: 420 days). Conclusions: Treatment with ponatinib was tolerable at a dose of 30mg qd with a toxicity profile comparable to other TKIs used in GIST. The majority of grade 3/4 AEs were hypertension or asymptomatic increases of laboratory values and thromboembolic events were rare. In a heavily pretreated patient population that lacks alternative treatment options the clinical activity was notable. An updated analysis including predictive biomarkers will be presented. Clinical trial information: NCT03171389 .

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Hanke, P. D., and R. V. Storti. "The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing." Molecular and Cellular Biology 8, no. 9 (September 1988): 3591–602. http://dx.doi.org/10.1128/mcb.8.9.3591-3602.1988.

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The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

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Hanke, P. D., and R. V. Storti. "The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing." Molecular and Cellular Biology 8, no. 9 (September 1988): 3591–602. http://dx.doi.org/10.1128/mcb.8.9.3591.

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The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

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Martinez Gomez, Laura, Fernando Pozo, Thomas A. Walsh, Federico Abascal, and Michael L. Tress. "The clinical importance of tandem exon duplication-derived substitutions." Nucleic Acids Research 49, no. 14 (July 24, 2021): 8232–46. http://dx.doi.org/10.1093/nar/gkab623.

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Abstract Most coding genes in the human genome are annotated with multiple alternative transcripts. However, clear evidence for the functional relevance of the protein isoforms produced by these alternative transcripts is often hard to find. Alternative isoforms generated from tandem exon duplication-derived substitutions are an exception. These splice events are rare, but have important functional consequences. Here, we have catalogued the 236 tandem exon duplication-derived substitutions annotated in the GENCODE human reference set. We find that more than 90% of the events have a last common ancestor in teleost fish, so are at least 425 million years old, and twenty-one can be traced back to the Bilateria clade. Alternative isoforms generated from tandem exon duplication-derived substitutions also have significantly more clinical impact than other alternative isoforms. Tandem exon duplication-derived substitutions have >25 times as many pathogenic and likely pathogenic mutations as other alternative events. Tandem exon duplication-derived substitutions appear to have vital functional roles in the cell and may have played a prominent part in metazoan evolution.

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Hampson, R. K., L. La Follette, and F. M. Rottman. "Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences." Molecular and Cellular Biology 9, no. 4 (April 1989): 1604–10. http://dx.doi.org/10.1128/mcb.9.4.1604-1610.1989.

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In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.

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Hampson, R. K., L. La Follette, and F. M. Rottman. "Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences." Molecular and Cellular Biology 9, no. 4 (April 1989): 1604–10. http://dx.doi.org/10.1128/mcb.9.4.1604.

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In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.

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del Arco, Araceli. "Novel variants of human SCaMC-3, an isoform of the ATP-Mg/Pi mitochondrial carrier, generated by alternative splicing from 3′-flanking transposable elements." Biochemical Journal 389, no. 3 (July 26, 2005): 647–55. http://dx.doi.org/10.1042/bj20050283.

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CaMCs (calcium-dependent mitochondrial carriers) represent a novel subfamily of metabolite carriers of mitochondria. The ATP-Mg/Pi co-transporter, functionally characterized more than 20 years ago, has been identified to be a CaMC member. There are three isoforms of the ATP-Mg/Pi carrier in mammals, SCaMC-1 (short CaMC-1), -2 and -3 (or APC-1, -3 and -2 respectively), corresponding to the genes SLC25A24, SLC25A25 and SLC25A23 respectively, as well as six N-terminal variants generated by alternative splicing for SCaMC-1 and -2 isoforms. In the present study, we describe four new variants of human SCaMC-3 generated by alternative splicing. The new mRNAs use the exon 9 3′-donor site and distinct 5′-acceptor sites from repetitive elements, in regions downstream of exon 10, the last exon in all SCaMCs. Transcripts lacking exon 10 (SCaMC-3b, -3b′, -3c and -3d) code for shortened proteins lacking the last transmembrane domain of 422, 456 and 435 amino acids, and were found in human tissues and HEK-293T cells. Mitochondrial targeting of overexpressed SCaMC-3 variants is incomplete. Surprisingly, the import impairment is overcome by removing the N-terminal extension of these proteins, suggesting that the hydrophilic N-terminal domain also participates in the mitochondrial import process, as shown for the CaMC members aralar and citrin [Roesch, Hynds, Varga, Tranebjaerg and Koehler (2004) Hum. Mol. Genet. 13, 2101–2111].

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Ren, Kehan, Zongjun Xia, Ermin Li, Xu Han, and Peng Ji. "Rapid Degradation of mDia2 Protein during Terminal Erythropoiesis Via an In Vivo Aid System: An Alternative Approach for Loss-of-Function Studies." Blood 142, Supplement 1 (November 28, 2023): 2443. http://dx.doi.org/10.1182/blood-2023-178268.

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Loss-of-function manipulations are crucial methods for studying gene functions. Despite the availability of many chemical-induced genetic manipulation techniques, there are limitations associated with these technologies. Tamoxifen-induced conditional gene expression, gene knockdown, or cell tracking represent some of the most common genetic manipulations employed in mice. However, tamoxifen-induced gene or Cre expression in bone marrow cell populations is relatively weak, affecting only a low proportion of cells. Similar observations have been made with the in vivo doxycycline-inducible system. Given that inflammation could affect the dispersion of bone marrow hematopoietic cells, an inducible system based on the MX1 promoter and interferon-alpha is not suitable for long-term protein knockdown. In addition, the above-mentioned methods are not able to achieve rapid loss-of-function because it is largely determined by the half-life of the transcriptional and translational products of the targeted gene. Furthermore, none of these technologies could achieve reversible depletion of the protein of interest. Therefore, there is a need for enhanced techniques for inducible and reversible loss-of-function gene manipulations in the field of hematopoiesis. The capacity for quick, targeted protein elimination is particularly necessary when researching the functions of proteins that play roles in swift biological processes. The Auxin-inducible degron (AID) system is a chemically inducible tool, widely employed for targeted protein degradation to study protein functions in cultured mammalian cells. It holds significant potential as a powerful tool for studying protein functions in vivo within the bone marrow. The AID system comprises three components: the auxin signal, AID tag, and auxin receptor F-box proteins (AFBs). When an AID tag is fused to a protein of interest, the AFB proteins recognize the tag and, in the presence of the auxin signal, induce the protein's degradation via the proteasome. Additionally, due to its small molecular weight, auxin can potentially penetrate the bone marrow barrier and achieve the working concentration in the bone marrow, facilitating protein degradation. In this study, we aim to investigate the role of mDia2 in terminal erythropoiesis using AID system. The mDia family proteins are formin proteins that catalyze the assembly of F-actin. The loss of function of mDia2 achieved by Cre-flox system leads to deficiency in enucleation and anemia. We hypothesize that the mDia2 loss of function could be compensated by upregulated mDia1 in the Cre-flox mediated knockout system, and a rapid loss of mDia2 protein could lead to more severe phenotypes in vivo and ex-vivo. We knocked in minilAA7-eGFP to the end of the last exon of mDia2 gene to create an endogenous tagged mDia2 protein in C57/B6 mice. We also created an AFB protein transgenic mouse by knocking in the atAFB2 protein coding gene at the ROSA26 locus driven by the Vav1 promoter. After crossing the mDia2-minilAA7-eGFP transgenic mice with Vav1-atAFB2 mice, we obtained mDia2-minilAA7-eGFP and Vav1-atAFB2 double homozygous mice. We administered auxin via intraperitoneal (IP) injection at a concentration of 300 mg/kg and collected total bone marrow cells four hours post-injection. Quantification of the GFP signal through flow cytometry revealed a significant reduction following auxin administration compared to the control group. This protein degradation was further confirmed with Western blotting assays. To understand how an immediate loss of mDia2 influences terminal erythropoiesis, we isolated lineage-negative cells from auxin-administered mice and cultured them in an EPO-containing medium with 10 µM auxin for 48 hours. Compared to lineage-negative cells from VavCre-mDia2f/f mice, auxin-induced mDia2 degradation resulted in a significantly lower enucleation rate. Notably, auxin administration had no observable effects on either WT mice or atAFB2 homozygous mice. This suggests that rapid mDia2 loss, induced by the AID system, permits a better understanding of the targeted protein's function by avoiding potential compensatory effects from mDia1 during terminal enucleation. In conclusion, our established in vivo AID system enables rapid targeted protein degradation and serves as a powerful tool for studying protein functions potentially obscured by compensatory effects during erythropoiesis.

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Libri, D., M. Goux-Pelletan, E. Brody, and M. Y. Fiszman. "Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene." Molecular and Cellular Biology 10, no. 10 (October 1990): 5036–46. http://dx.doi.org/10.1128/mcb.10.10.5036-5046.1990.

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The beta tropomyosin gene contains two internal exons which are spliced in a mutually exclusive manner. Exon 6B is specifically included in the mature transcripts expressed in skeletal muscle or cultured myotubes, while exon 6A is a myoblast- or smooth muscle-specific exon. The intron between them, which is never spliced in normal conditions, contains two characteristic features: first, the unusual location of the branch point at position -105 from the acceptor, and second, the presence of a very long pyrimidine stretch upstream of the skeletal muscle exon. In this study we designed a number of sequence modifications to investigate the role of these two elements and of a computer-predicted secondary structure in the mutually exclusive splicing of the two exons. We found that mutations in the skeletal exon as well as in the upstream intron could change in vivo the tissue-specific pattern as well as the mutually exclusive character of the two exons. Our results suggest that the unusual position of the branch point does not prevent the utilization of exon 6B in myoblasts and that the region around the acceptor site of exon 6B and the polypyrimidine tract have an important role in this control. Last, we discuss the possible implications of secondary structures.

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Libri, D., M. Goux-Pelletan, E. Brody, and M. Y. Fiszman. "Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene." Molecular and Cellular Biology 10, no. 10 (October 1990): 5036–46. http://dx.doi.org/10.1128/mcb.10.10.5036.

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The beta tropomyosin gene contains two internal exons which are spliced in a mutually exclusive manner. Exon 6B is specifically included in the mature transcripts expressed in skeletal muscle or cultured myotubes, while exon 6A is a myoblast- or smooth muscle-specific exon. The intron between them, which is never spliced in normal conditions, contains two characteristic features: first, the unusual location of the branch point at position -105 from the acceptor, and second, the presence of a very long pyrimidine stretch upstream of the skeletal muscle exon. In this study we designed a number of sequence modifications to investigate the role of these two elements and of a computer-predicted secondary structure in the mutually exclusive splicing of the two exons. We found that mutations in the skeletal exon as well as in the upstream intron could change in vivo the tissue-specific pattern as well as the mutually exclusive character of the two exons. Our results suggest that the unusual position of the branch point does not prevent the utilization of exon 6B in myoblasts and that the region around the acceptor site of exon 6B and the polypyrimidine tract have an important role in this control. Last, we discuss the possible implications of secondary structures.

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Joiner, Clinton H., Scott Crable, and Patrick G. Gallagher. "Alternative Splicing within Exon 1 of the KCl Cotransporter-3 (KCC3) Gene Results in Novel Transcripts in Erythroid Cells." Blood 108, no. 11 (November 16, 2006): 1564. http://dx.doi.org/10.1182/blood.v108.11.1564.1564.

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Abstract The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, including KCC3, are expressed in erythroid cells (Exp.Hematol.2005;33:624), but their relative contribution to KCC fluxes and volume regulation in red cells remains unknown. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1646 bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 5 different transcripts. Three major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 103bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of the exon, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (KCC3a-Short of Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains the first 668bp of the exon (including both exons 1D and 1C) as 5′ untranslated sequence, then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of ‘full-length’ KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Reporter activity increased 3 fold upon removal of the 590 bp 3′ segment of such constructs, suggesting the presence of negative regulatory elements within the exon immediately upstream of exon 1A. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.

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Tien, Jerry F., Alborz Mazloomian, S. W. Grace Cheng, Christopher S. Hughes, Christalle C. T. Chow, Leanna T. Canapi, Arusha Oloumi, et al. "CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion." Nucleic Acids Research 45, no. 11 (March 17, 2017): 6698–716. http://dx.doi.org/10.1093/nar/gkx187.

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21

Costas, María Jesús, Ana Couto, Alicia Cabezas, Rosa María Pinto, João Meireles Ribeiro, and José Carlos Cameselle. "Alternative Splicing of the Last TKFC Intron Yields Transcripts Differentially Expressed in Human Tissues That Code In Vitro for a Protein Devoid of Triokinase and FMN Cyclase Activity." Biomolecules 14, no. 10 (October 12, 2024): 1288. http://dx.doi.org/10.3390/biom14101288.

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The 18-exon human TKFC gene codes for dual-activity triokinase and FMN cyclase (TKFC) in an ORF, spanning from exon 2 to exon 18. In addition to TKFC-coding transcripts (classified as tkfc type by their intron-17 splice), databases contain evidence for alternative TKFC transcripts, but none of them has been expressed, studied, and reported in the literature. A novel full-ORF transcript was cloned from brain cDNA and sequenced (accession no. DQ344550). It results from an alternative 3′ splice-site in intron 17. The cloned cDNA contains an ORF also spanning from exon 2 to exon 18 of the TKFC gene but with a 56-nt insertion between exons 17 and 18 (classified as tkfc_ins56 type). This insertion introduces an in-frame stop, and the resulting ORF codes for a shorter TKFC variant, which, after expression, is enzymatically inactive. TKFC intron-17 splicing was found to be differentially expressed in human tissues. In a multiple-tissue northern blot using oligonucleotide probes, the liver showed a strong expression of the tkfc-like splice of intron 17, and the heart preferentially expressed the tkfc_ins56-like splice. Through a comparison to global expression data from massive-expression studies of human tissues, it was inferred that the intestine preferentially expresses TKFC transcripts that contain neither of those splices. An analysis of transcript levels quantified by RNA-Seq in the GTEX database revealed an exception to this picture due to the occurrence of a non-coding short transcript with a tkfc-like splice. Altogether, the results support the occurrence of potentially relevant transcript variants of the TKFC gene, differentially expressed in human tissues. (This work is dedicated in memoriam to Professor Antonio Sillero, 1938–2024, for his lifelong mentoring and his pioneering work on triokinase).

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22

Sogawa, Chiharu, Kei Kumagai, Norio Sogawa, Katsuya Morita, Toshihiro Dohi, and Shigeo Kitayama. "C-terminal region regulates the functional expression of human noradrenaline transporter splice variants." Biochemical Journal 401, no. 1 (December 11, 2006): 185–95. http://dx.doi.org/10.1042/bj20060495.

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The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

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Hu, Zhigang, Junting Cao, Jianqin Zhang, Liyan Ge, Huilin Zhang, and Xiaolin Liu. "Skeletal Muscle Transcriptome Analysis of Hanzhong Ma Duck at Different Growth Stages Using RNA-Seq." Biomolecules 11, no. 2 (February 19, 2021): 315. http://dx.doi.org/10.3390/biom11020315.

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As one of the most important poultry worldwide, ducks (Anas platyrhynchos) are raised mainly for meat and egg products, and muscle development in ducks is important for meat production. Therefore, an investigation of gene expression in duck skeletal muscle would significantly contribute to our understanding of muscle development. In this study, twenty-four cDNA libraries were constructed from breast and leg muscles of Hanzhong Ma ducks at day 17, 21, 27 of the embryo and postnatal at 6-month-old. High-throughput sequencing and bioinformatics were used to determine the abundances and characteristics of transcripts. A total of 632,172,628 (average 52,681,052) and 637,213,938 (average 53,101,162) reads were obtained from the sequencing data of breast and leg muscles, respectively. Over 71.63% and 77.36% of the reads could be mapped to the Anas platyrhynchos genome. In the skeletal muscle of Hanzhong duck, intron variant (INTRON), synonymous variant (SYNONYMOUS_CODING), and prime 3′ UTR variant (UTR_3_PRIME) were the main single nucleotide polymorphisms (SNP) annotation information, and “INTRON”, “UTR_3_PRIME”, and downstream-gene variant (DOWNSTREAM) were the main insertion-deletion (InDel) annotation information. The predicted number of alternative splicing (AS) in all samples were mainly alternative 5′ first exon (transcription start site)-the first exon splicing (TSS) and alternative 3′ last exon (transcription terminal site)-the last exon splicing (TTS). Besides, there were 292 to 2801 annotated differentially expressed genes (DEGs) in breast muscle and 304 to 1950 annotated DEGs in leg muscle from different databases. It is worth noting that 75 DEGs in breast muscle and 49 DEGs in leg muscle were co-expressed at all developmental points of comparison, respectively. The RNA-Seq data were confirmed to be reliable by qPCR. The identified DEGs, such as CREBL2, RHEB, GDF6, SHISA2, MYLK2, ACTN3, RYR3, and STMN1, were specially highlighted, indicating their strong associations with muscle development in the Hanzhong Ma duck. KEGG pathway analysis suggested that regulation of actin cytoskeleton, oxidative phosphorylation, and focal adhesion were involved in the development of skeletal muscle. The findings from this study can contribute to future investigations of the growth and development mechanism in duck skeletal muscle.

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Crable, Scott, Clinton H. Joiner, and Patrick G. Gallagher. "A GC Box Element Is Critical for Transcriptional Regulation of the K-Cl Cotransporter Isoform KCC3a in Hematopoetic Cells." Blood 110, no. 11 (November 16, 2007): 1712. http://dx.doi.org/10.1182/blood.v110.11.1712.1712.

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Abstract The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, are expressed in erythroid cells (Crable et al. Exp. Hematol.2005;33:624). Although the relative contribution of the three KCC isoforms to KCC fluxes and volume regulation in red cells remains unknown, KCC3 appears to be the dominant transcript in late erythroid cells. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1718bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 10 different transcripts. Six major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 167bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of exon 1, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (called KCC3a-Short by Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains 629bp of the exon immediately 3′ of exon 1D as 5′ untranslated sequence (exon 1C), then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of full-length KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Further deletion analysis shows that a minimal promoter containing less than 125 base pairs yields full promoter activity. A mutation of a GC box within this region reduces activity over 15 fold, implicating SP-1 as a trans-regulatory factor. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional and translational control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.

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Carrion, Shane A., Jennifer J. Michal, and Zhihua Jiang. "Alternative Transcripts Diversify Genome Function for Phenome Relevance to Health and Diseases." Genes 14, no. 11 (November 8, 2023): 2051. http://dx.doi.org/10.3390/genes14112051.

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Manipulation using alternative exon splicing (AES), alternative transcription start (ATS), and alternative polyadenylation (APA) sites are key to transcript diversity underlying health and disease. All three are pervasive in organisms, present in at least 50% of human protein-coding genes. In fact, ATS and APA site use has the highest impact on protein identity, with their ability to alter which first and last exons are utilized as well as impacting stability and translation efficiency. These RNA variants have been shown to be highly specific, both in tissue type and stage, with demonstrated importance to cell proliferation, differentiation and the transition from fetal to adult cells. While alternative exon splicing has a limited effect on protein identity, its ubiquity highlights the importance of these minor alterations, which can alter other features such as localization. The three processes are also highly interwoven, with overlapping, complementary, and competing factors, RNA polymerase II and its CTD (C-terminal domain) chief among them. Their role in development means dysregulation leads to a wide variety of disorders and cancers, with some forms of disease disproportionately affected by specific mechanisms (AES, ATS, or APA). Challenges associated with the genome-wide profiling of RNA variants and their potential solutions are also discussed in this review.

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Essand, M., S. Vikman, J. Grawé, L. Gedda, C. Hellberg, K. Oberg, T. H. Totterman, and V. Giandomenico. "Identification and characterization of a novel splicing variant of vesicular monoamine transporter 1." Journal of Molecular Endocrinology 35, no. 3 (December 2005): 489–501. http://dx.doi.org/10.1677/jme.1.01875.

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Vesicular monoamine transporter 1 (VMAT1) is an integral protein in the membrane of secretory vesicles of neuroendocrine and endocrine cells that allows the transport of biogenic monoamines, such as serotonin, from the cytoplasm into the secretory vesicles. The full-length VMAT1 transcript is produced from 16 exons. We have identified and characterized an alternatively spliced form of VMAT1 that lacks exon 15, the next to last exon of VMAT1. The new form was therefore denoted VMAT1Δ15. Exon 15 does not contain an even multiple of three nucleotides. As a consequence, there is a shift of reading frame, and exon 16 is translated in an alternative reading frame, yielding a novel protein with a shorter and unrelated C-terminus compared with the native VMAT1 protein. VMAT1 and VMAT1Δ15 mRNAs are simultaneously expressed in normal and neoplastic neuroendocrine cells of the GI tract. However, VMAT1 expression is always higher than VMAT1Δ15 expression. We prove that VMAT1Δ15 is not localized in large, dense core vesicles as the native form but in the endoplasmic reticulum. Furthermore, while VMAT1 can take up serotonin, VMAT1Δ15 cannot, indicating different functions for the two forms of VMAT1.

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Sumanasekera, Chiranthani, David S. Watt, and Stefan Stamm. "Substances that can change alternative splice-site selection." Biochemical Society Transactions 36, no. 3 (May 21, 2008): 483–90. http://dx.doi.org/10.1042/bst0360483.

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Alternative pre-mRNA splicing is an important element in eukaryotic gene expression, as most of the protein-coding genes use this process to generate multiple protein isoforms from a single gene. An increasing number of human diseases are now recognized to be caused by the selection of ‘wrong’ alternative exons. Research during the last few years identified a number of low–molecular-mass chemical substances that can change alternative exon usage. Most of these substances act by either blocking histone deacetylases or by interfering with the phosphorylation of splicing factors. How the remaining large number of these substances affect splicing is not yet fully understood. The emergence of these low-molecular-mass substances provides not only probes for studying alternative pre-mRNA splicing, but also opens the door to the possible harnessing of these compounds as drugs to control diseases caused by the selection of ‘wrong’ splice sites.

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28

Duthie, S. M., P. L. Taylor, and K. A. Eidne. "Characterization of the mouse thyrotrophin-releasing hormone receptor gene: an exon corresponds to a deletion in the rat cDNA." Journal of Molecular Endocrinology 11, no. 2 (October 1993): 141–49. http://dx.doi.org/10.1677/jme.0.0110141.

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ABSTRACT The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5′ untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3′ UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3′ UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3′ UTR, allowing transcription to continue into the 3′ UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA. This would then result in an alternative 412 amino acid version of the mouse TRH-R protein, with 95% homology to the rat TRH-R. This study focused on the structural differences in the intracellular COOH-terminal tail of the receptor, which is known to be a functionally important domain in other members of the G protein-coupled receptor family. We have also recently characterized the human TRH-R cDNA, which revealed a third variant at the COOH terminus. Comparisons between mouse, rat and human TRH-Rs show that the amino acid sequences are virtually identical. However, significant differences between these species exist at the COOH terminus, with each TRH-R having a unique form of the COOH-terminal tail, beginning at exactly the same site and encoding 1, 20 and 6 amino acids in the mouse, rat and human respectively.

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Santoro, Alessandra, Lea Dagnino, Cecilia Agueli, Domenico Salemi, Maria Grazia Bica, Annalisa Marfia, Sonia Cannella, et al. "Altered mRNA Expression of Pax-5 Is a Common Event in Acute Lymphoblastic Leukemia." Blood 112, no. 11 (November 16, 2008): 1189. http://dx.doi.org/10.1182/blood.v112.11.1189.1189.

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Abstract Introduction. Pax-5 gene codifies for a transcription factor central to B-cell differentiation and function, expressed during the early stage of differentiation and alternatively spliced during B-cell development. In addition to the full-length isoform (Pax-5a), isoforms arising from the inclusion or exclusion of exons 2, 7, 8, and/or 9, and lacking the DNA-binding and the transactivating domains, have been described. These isoforms and their levels of expression increase during B-cell maturation. In particular, Pax-5b isoform (deleted of exon 2), resulting in protein with partial DNA-binding domain, is related with the differentiation stage. Recently, mutations of Pax-5 gene were reported in 31.7% of 242 children (Mullighan, Nature 2007) and 30% of 119 adults (Familiades, ASH 2007 abs. 2806) with B-cell precursor acute lymphoblastic leukemia (ALL). We investigated Pax-5 gene mutations and isoforms expression in patients with ALL at the diagnosis and in selected cell populations from control subjects. Methods. Pax-5 mutations and mRNA isoforms were investigated by sequence analysis. Pax-5a and Pax-5b expression levels were evaluated by quantitative PCR in leukemic cells. Total RNA was obtained from leukemic blasts of 95 patients with B-cell precursor ALL at diagnosis, 45 adults and 50 children. These last had been selected according to the presence of adverse translocations: BCR/ABL p190 (n=10), E2A-PBX1 (n=10), TEL/AML (n=10), MLL/AF4 (n=10), or none (n=10). mRNA was obtained from purified peripheral blood CD19+ lymphocytes (6 samples), from bone marrow CD34+ hematopoietic progenitor cells (6 samples), and pro-B CD34+CD19+ cells (3 samples) from a total of 8 healthy bone marrow donors. Results. Pax-5 gene point mutations were found in 8/45 adults (18%) and 14/50 children (28%) (p=n.s.), resulting in a total of 20 different mutations. Mutations were located as follows: exon 2 (53G>C, 76delG, 101C>G, 113G>A, 197G>A), exon 3 (223A>G, 247delA, 296T>C), exon 4 (446A>T, 526G>A), exon 5 (580A>C, 601G>A), exon 6 (625C>T, 716G>A), exon 7 (836C>A), exon 8 (955G>A, 964insC, 971A>G), exon 9 (1058C>T), exon 10 (1133G>A). There was no correlation between the presence of mutations and the listed genetic subtypes. Pax-5 alternative splicing was observed in 47/95 cases (49%): 29/45 adults (64%) and 18/50 children (36%) (p=n.s.). Alternative splicing resulted in increased frequencies of the Pax-5b isoform and the deletion of exons 8 and/or 9; furthermore, a novel isoform resulting from the skipping of exon 5 was documented. On the contrary, only the fulllenght isoform was present in CD34+ progenitors and CD34+CD19+ cells from healthy donors, in which deleted isoforms were detected only at the stage of mature B-lymphocytes. Conclusion: Pax-5 mutations are frequent in childhood and adult ALL and are scattered all over the gene. We extend the current knowledge on their pathogenic role by demonstrating that alternative splicing is a common event in these patients. Imbalance between the fulllength and the Pax-5b isoforms is expected to affect the maturation of the B-cell and its propensity to apoptosis (Robichaud Nucleic Acids Res 2008), thus contributing to the process of leukemogenesis. Based on our findings, this mechanism acts broadly and is independent of the most common ALL genetic translocations.

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Catarsi, Paolo, Vittorio Rosti, Vittorio Abbonante, Alessandra Balduini, Gaetano Bergamaschi, Elisa Bonetti, Valentina Poletto, Laura Villani, and Giovanni Barosi. "JAK2 exon 14 Skipping in Patients with Primary Myelofibrosis (PMF),." Blood 118, no. 21 (November 18, 2011): 3844. http://dx.doi.org/10.1182/blood.v118.21.3844.3844.

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Abstract Abstract 3844 In recent years the diagnosis and treatment of patients with myeloproliferative disorders has focused on the JAK2 gene. Recently, an alternative splicing of JAK2 transcript lacking the entire exon 14 (88bp), has been described (Ma et al. (2010) PLoS ONE, 5, e12165). This mRNA has a stop codon in exon 15 and is translated into a truncated protein that has a deletion within the JH2 pseudokinase domain and complete deletion of the kinase domain (JH1). The alternative transcript was detected in plasma of patients with myeloproliferative neoplasms (MPNs) (58% of patients positive for the V617F mutation and 33% of negative) in proportions ranging from 2% to 26% compared to the normal transcript, but never in healthy subjects. However, quantification of the transcript was performed by quantitative fragment length analysis, which is not the most appropriate method for such determination especially if, as in this case, the PCR is not limited to the exponential phase (Cirigliano, V. et al. (2001) Mol. Hum. Reprod., 7, 1001–1006). We investigate the mRNA levels of JAK2 and its splicing variant lacking exon 14, in granulocytes isolated from peripheral blood of 9 healthy individuals and 41 patients diagnosed with primary myelofibrosis (19 negative and 22 positive for the JAK2 V617F mutation). To overcome the limitations of quantitative fragment length analysis, the levels of the splice variant were assessed by qPCR, using a boundary-spanning primer designed on the junction between exons 13 and 15 (Vandenbroucke II, et al. (2001) Nucleic Acids Res., 29:E68). The JAK2 V617F allele burden was measured as previously described (Lippert, E. et al. (2006) Blood, 108, 1865–1867). Low levels of the alternative-spliced product was found in all RNA samples from patients and from all healthy individuals, in proportions ranging from 0.3 % to 3.5% compared to the normal transcript. The wild-type patients (95% C.I.: 0.3–0.8%) did not differ from controls (95% C.I.: 0.4–0.7%) while, in patients with the JAK2 V617F mutation, the levels correlates with allele burden (R^2= 0.513; p= 0.0002) (Figure 1).Figure 1Figure 1. To explain this observation we performed a bioinformatic analysis of the exon 14 sequence, using the software Human Splice Finder 2.4.1 (Desmet, F.O. et al. (2009) Nucleic Acids Res37 (e67) and ESE finder 3.0 (Cartegni, L. et al. (2003) Nucleic Acids Res31 (13)). The G/T transversion causing the aminoacidic substitution V617F also breaks an high-score, putative, Exon Splicing Enhancer (ESE) sequence that potentially binds the splicing regulator protein SRP55 (TGTGTC; positions 70 to 75 of exon 14). The higher level of the splice variant found in patients with the mutation could be due to an alteration of the binding of molecular factors that determine the inclusion of exon 14 in mature transcripts. The low levels of alternative transcripts observed, could be due to the presence of a stop codon in exon 15 as a result of the frameshift caused by the exclusion of exon 14. We know that a stop codon at more than 55 bases upstream of the last exon junction (in this case between exons 24 and 25) determines the activation of the Nonsense Mediated mRNA Decay (NMD) system and the consequent destruction of the messenger. In order to knowhow a small percentage of mRNA, subjected to degradation by NMD system, can produce the predicted 70 KDa protein that has been detected after immunoprecipitation and western blot analysis using JAK2 N-terminal antibodies, we are characterizing the 3' end of the alternative transcript. We show that the somatic mutation in exon 14 of JAK2 gene, in addition to the effects caused by the V617F amino acid change, has a moderate influence on splicing, increasing the levels of a splicing variant that is produced in small amounts also in healthy subjects. Our study demonstrates the efficacy of qPCR technique and the predictive power of bioinformatics tools used here. Further studies are needed to understand how this isoform is processed at the post-transcriptional level and whether there are significant effects on clinical phenotype. Disclosures: Barosi: Novartis: Membership on an entity's Board of Directors or advisory committees.

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Herbrechter, Robin, Nadine Hube, Raoul Buchholz, and Andreas Reiner. "Splicing and editing of ionotropic glutamate receptors: a comprehensive analysis based on human RNA-Seq data." Cellular and Molecular Life Sciences 78, no. 14 (June 8, 2021): 5605–30. http://dx.doi.org/10.1007/s00018-021-03865-z.

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AbstractIonotropic glutamate receptors (iGluRs) play key roles for signaling in the central nervous system. Alternative splicing and RNA editing are well-known mechanisms to increase iGluR diversity and to provide context-dependent regulation. Earlier work on isoform identification has focused on the analysis of cloned transcripts, mostly from rodents. We here set out to obtain a systematic overview of iGluR splicing and editing in human brain based on RNA-Seq data. Using data from two large-scale transcriptome studies, we established a workflow for the de novo identification and quantification of alternative splice and editing events. We detected all canonical iGluR splice junctions, assessed the abundance of alternative events described in the literature, and identified new splice events in AMPA, kainate, delta, and NMDA receptor subunits. Notable events include an abundant transcript encoding the GluA4 amino-terminal domain, GluA4-ATD, a novel C-terminal GluD1 (delta receptor 1) isoform, GluD1-b, and potentially new GluK4 and GluN2C isoforms. C-terminal GluN1 splicing may be controlled by inclusion of a cassette exon, which shows preference for one of the two acceptor sites in the last exon. Moreover, we identified alternative untranslated regions (UTRs) and species-specific differences in splicing. In contrast, editing in exonic iGluR regions appears to be mostly limited to ten previously described sites, two of which result in silent amino acid changes. Coupling of proximal editing/editing and editing/splice events occurs to variable degree. Overall, this analysis provides the first inventory of alternative splicing and editing in human brain iGluRs and provides the impetus for further transcriptome-based and functional investigations.

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Mamala, A., and W. Sciężor. "Evaluation of the Effect of Selected Alloying Elements on the Mechanical and Electrical Aluminium Properties." Archives of Metallurgy and Materials 59, no. 1 (March 1, 2014): 413–17. http://dx.doi.org/10.2478/amm-2014-0069.

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Abstract Modern industry expects aluminum products with new, unusual, and well-defined functional properties. Last years we are able to notice constant development of aluminium alloys. In food industry, power engineering, electrical engineering and building engineering, flat rolled products of 1XXX series aluminium alloys are used.8XXX series alloys registered in Aluminium Association during last 20 years may be used as an alternative. These alloys have very good thermal and electrical conductivity and perfect technological formability. Moreover, these materials are able to obtain by aluminium scrap recycling. Fundamental alloy additives of 8XXX series are Fe, Si, Mn, Mg, Cu and Zn. Aluminium alloying with these additives makes it possible to obtain materials with different mechanical ale electrical properties. In this paper, the analysis of alloy elements content (in 8XXX series) effect on chosen properties of material in as cast and after thermal treatment tempers has been presented.

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Gee, Sherry, Jonathan Villalobos, Miki Yamamoto, Tyson A. Clark, Jeong-Ah Kang, Amittha Wickrema, Joel Anne Chasis, and john G. Conboy. "Stage-Specific Switches in Alternative Pre-mRNA Splicing during Late Erythropoiesis Are Conserved from Mouse to Human." Blood 112, no. 11 (November 16, 2008): 531. http://dx.doi.org/10.1182/blood.v112.11.531.531.

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Abstract Spatial and temporal regulation of alternative pre-mRNA splicing determines which exons are incorporated into mature mRNA, modulating mRNA coding capacity to ensure synthesis of appropriate protein isoforms throughout normal differentiation and development. During erythropoiesis, a stage-specific switch in pre-mRNA splicing activates incorporation of protein 4.1R exon 16, thereby increasing 4.1R affinity for spectrin and actin and mechanically strengthening red blood cell membranes. We are exploring the hypothesis that stage-specific changes in pre-mRNA splicing regulate expression of other critical genes during terminal erythropoiesis. Last year we described exon microarray and RT-PCR studies that revealed several novel pre-mRNA splicing switches in terminally differentiating human erythroid progenitors. These alternative splicing events involved well-annotated exons with consensus exon-intron boundaries, supporting a model in which these events represent a regulated alternative splicing program rather than a breakdown of splicing integrity in late erythropoiesis. Here we report additional evidence for this model by showing that several erythroid stage-specific switches in alternative pre-mRNA splicing are conserved between human and mouse. Primary mouse splenic erythroblasts from FVA-infected mice were cultured in vitro under differentiation conditions and used as the source of RNA for analysis of murine erythroid splicing events. From a total of seven internal cassette exons whose splicing was activated in late human erythroblasts, five exhibited an analogous splicing switch in murine erythroblasts. Comparative genomic analysis showed that these alternative exons are embedded in regions of unusually high sequence conservation among vertebrate species, suggesting that important regulatory signals are contained within the adjacent introns. Indeed, the flanking introns for several of these exons contain binding motifs for Fox2, an RNA binding protein and known splicing regulator for many tissue-specific splicing events. Further analysis of the conserved erythroid splicing events revealed the following: three splicing switches occur in transcripts encoding RNA binding proteins (MBNL2, HNRPLL, and SNRP70), suggesting significant changes in the RNA processing machinery of late erythroblasts; and three of these alternative exons encode premature stop codons that could induce nonsense mediated decay (NMD) and contribute to down-regulation of these genes during terminal erythropoiesis. Consistent with the latter hypothesis, inhibition of NMD in murine erythroblast cultures led to increased accumulation of mRNA isoforms containing the premature stop codons. Together these results suggest the existence of a highly regulated alternative splicing program that is critical for late erythroid differentiation.

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Shen, Yulei, Baosheng Ge, Himabindu Ramachandrareddy, Timothy McKeithan, and Wing-Chung Chan. "Alternative Splicing Generates a BCL6 Isoform Encoding a Compact Repressor." Blood 108, no. 11 (November 1, 2006): 2383. http://dx.doi.org/10.1182/blood.v108.11.2383.2383.

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Abstract BCL6 encodes a BTB/POZ zinc finger (ZF) transcriptional repressor which is essential for normal germinal center (GC) reactions. Its dysregulated expression contributes to the pathogenesis of B cell non-Hodgkin lymphoma (NHL). The BCL6 gene consists of ten exons, BCL6 translation start site is located in exon 3, and the six-ZF DNA binding domain is encoded by exons 7 to 10. BCL6S, was cloned from the cDNA of a BCL-6 positive cell line, DHL-16. DNA sequencing showed that BCL6S was a normal splicing isoform that excludes the entire exon 7 of the BCL6 gene and encodes a 650 amino acid (aa) protein. BCL6S was detected by RT-PCR in all human cell lines and tissues expressing BCL6, but was not detected in the mouse B cell lymphoma cell line A20. BCL6S accounts for 1/8 to 1/10 of total BCL6 transcripts. Luciferase reporter assays demonstrated that BCL6S could repress typical BCL6 target genes ( BCL6, CD23b, Blimp1, MIP1α ) as effectively as the full length BCL6. BCL6S retains the last four ZFs of BCL6 and localizes in the cell nucleus. Protein-protein interaction assays confirmed that BCL6 and BCL6S could form homodimers and heterodimers. DNA binding assays showed no affinity differences between BCL6 and BCL6S; however, a single mutation at the N-terminus of the POZ domain (L19 to H) not only disrupted BCL6S dimerization but also significantly decreased the DNA binding affinity, indicating that BCL6 dimerization may stabilize the ZF-DNA binding. In conclusion, we have identified a novel BCL6 splicing isoform. BCL6S, a compact repressor as potent as BCL6. POZ dimerization plays an important role in stabilization of BCL6 DNA binding whereas the first two ZFs are not absolutely required. The biological role of BCL6S in GC B cells needs to be further investigated.

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Razavian, Niema, and Vivian Cheung. "SRSF1 Is a Mediator of Radiation-Induced Alternative Splicing in B-Lymphocytes." Blood 128, no. 22 (December 2, 2016): 1341. http://dx.doi.org/10.1182/blood.v128.22.1341.1341.

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Abstract Ionizing radiation is used in the treatment of Hodgkin and non-Hodgkin lymphomas (Spetch et al. 2014; Illidge et al. 2014). Despite its effectiveness, radiation is a "blunt" therapy that damages indiscriminately both cancer and normal cells, and can result in secondary malignancies (Dores et al. 2002). To better understand cellular response to radiation, we examined alternative splicing, and its regulation, in irradiated human cells. To accomplish this, we exposed cultured B-lymphocytes from 10 individuals to 10 Gy of ionizing radiation, and performed RNA sequencing before, and two and six hours after radiation treatment. With these data, we first identified alternative splicing events. From about 60 million reads per sample, we detected over 20,000 alternatively spliced events. In total, 1,600 events were radiation responsive (ANOVA, FDR<5%). The splicing events in irradiated cells belong mainly to three categories, each of which occurred in genes with distinct biological functions. Cassette exons, which were the most numerous splicing event, were found primarily in DNA damage response and apoptotic genes, while alternative first or last exons were found in chromatin assembly genes; retained introns occurred in genes involved in RNA processing and translation. Using these radiation-responsive events, we then examined the kinetics of this response. We found that changes in alternative splicing were acute, occurring within two hours of radiation treatment. Overall, the splicing events produced shorter transcripts in irradiated cells. Next, we looked for how alternative splicing is regulated in response to radiation. To identify putative mediators of this response, we determined the expression levels of over 60 trans-acting splicing factors. We found that the expression level of 26 splicing factors changed significantly in irradiated cells (ANOVA, FDR<5%). Of these putative mediators, we further examined serine/arginine-rich splicing factor (SRSF1)'s role in radiation response. First, we assessed SRSF1 expression in irradiated cells. Following radiation exposure, the transcript and protein expressions of SRSF1 decreased. Next, we performed motif enrichment analysis to identify target genes. SRSF1 RNA-binding sites were enriched in skipped cassette exons: specifically, of the 362 skipped cassette exons in irradiated cells, 93 (26%) contained SRSF1 binding motifs. Finally, we examined the link between SRSF1 and exon skipping in irradiated cells more closely. For example, in the transcripts for RNA-binding protein 3 (RBM3), SRSF1 binds an RBM3 exon that contains a premature termination codon (Sanford et al. 2009), and mediates exclusion of this exon. We confirmed experimentally that this results in an increase in RBM3 protein expression. Together, our data demonstrate that alternative splicing is a key part of cellular response to radiation, and SRSF1 plays a role in mediating exon skipping. In this presentation, we will describe radiation-induced alternative splicing by discussing the genes that are alternatively spliced, and its regulation by the splicing factor SRSF1. Disclosures No relevant conflicts of interest to declare.

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Tabata, Hidemi, Momoko Kobayashi, Junko H. Ikeda, Nobuhiro Nakao, Toru R. Saito, and Minoru Tanaka. "Characterization of multiple first exons in murine prolactin receptor gene and the effect of prolactin on their expression in the choroid plexus." Journal of Molecular Endocrinology 48, no. 2 (January 31, 2012): 169–76. http://dx.doi.org/10.1530/jme-11-0122.

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Prolactin (Prl) receptor (Prlr) gene is expressed in various brain regions, with the highest level present in the choroid plexus, a site for receptor-mediated PRL transport from the blood to cerebrospinal fluid. We investigated the regulatory mechanism ofPrlrgene expression by PRL in the murine choroid plexus. We first examined the organization of the alternative first exons in murinePrlrgene. In addition to the three known first exons, mE11, mE12, and mE13, two first exons, mE14and mE15, were newly identified by cDNA cloning. Each first exon variant ofPrlrmRNA exhibited tissue-specific or generic expression. In the choroid plexus of mice, the expression levels ofmE13-,mE14-, andmE15-PrlrmRNAs were increased in the lactating mice compared with those in the diestrus mice. Furthermore, the expression level ofmE14-PrlrmRNA was decreased in the PRL-deficient (Prl−/−) mice compared with the PRL-normal (Prl+/+andPrl+/−) mice. In the ovariectomizedPrl−/−mice, the expression level ofmE14-PrlrmRNA was significantly increased by PRL administration but not by 17β-estradiol administration. The expression levels of the two last exon variants ofPrlrmRNAs, encoding the long and short cytoplasmic regions of PRLR, were also increased in the lactating mice and decreased in thePrl−/−mice. These findings suggest that PRL stimulates thePrlrgene expression through the transcriptional activation of mE14first exon, leading to increases in the long- and short-form variants ofPrlrmRNA in the murine choroid plexus.

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Kuwahara, Mitsuhiro, Sumiko Kurachi, and Kotoku Kurachi. "Molecular Mechanism of Prothrombin G20210A Variant: Critical New Role of Exon Splicing Enhancer in Poly (A) Tailing." Blood 104, no. 11 (November 16, 2004): 1944. http://dx.doi.org/10.1182/blood.v104.11.1944.1944.

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Abstract Although prothrombin G20210A variant was first found in 1996 as a genetic risk factor of thrombosis, the underlying molecular mechanism for elevation of circulatory prothrombin levels due to the variant has remained elusive. The position 20210 is at the poly (A) tailing site, and several research groups reported different, contradictive possibilities including enhancement in poly (A) tailing, change in RNA stability or no difference in prothrombin mRNA level between G (wild type) and A (variant) at position 20210. Such confusions appear to be due to no reliable assay model availability. To overcome these problems, we developed a minigene assay system composed of only autologous components of human prothrombin gene, including its promoter, coding regions, introns and 3′UTR with poly (A) signal. A minigene containing the first intron and the last intron did not express at any appreciable levels, failing proper splicing of the last intron. Inclusion of upstream other introns was found essential for its appropriate splicing. Although it is generally accepted that the last intron enhances the efficiency of poly (A) tailing, we found that it is not the case for the prothrombin gene. Instead, an exon-splicing enhancer (ESE) of another upstream intron was found to be responsible for efficient poly (A) tailing. ESE, therefore, plays a critical role not only in alternative splicing but also in constitutive RNA processing, including poly (A) tailing. Etiology of prothrombin G20210A variant studied with culture cells and transgenic mice, supported an enhancement of poly (A) tailing, and not a change in mRNA stability.

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Renosi, Florian, Ambre Giguelay, Jean Francis Berry, Evan Seffar, Sabeha Biichlé, Alexis Overs, Xavier Roussel, et al. "Blastic Plasmacytoid Dendritic Cell Neoplasm Are Not Characterized By Two Distinct Subgroups, but By a Transcriptomic and Phenotypic Gradient." Blood 144, Supplement 1 (November 5, 2024): 6106. https://doi.org/10.1182/blood-2024-206941.

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Background: Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is a rare haematological malignancy characterized by NF-κB activation (Sapienza et al., 2014), but also BCL2 overexpression (Montero et al., 2017), UV exposure damages (Griffin et al., 2023), neural signatures (Sapienza et al., 2021) and fatty acid metabolism (Ceroi et al., 2016). However, BPDCN is heterogenous: recent studies showed two subgroups, based on immune response involving CD11b/CD177 (Summerer et al., 2021) or on pure pDC/conventional DC-enriched signatures (Künster et al., 2022). Künster et al. also showed NF-κB signalling pathway versus EZH2 dependence (Künster et al., 2024). Methods: We conducted a retrospective study on bone marrow (BM) aspirates and peripheral blood (PB) samples obtained at diagnosis (French pDC network ROMI DC-2008-713/DC-2016-27-91, Besançon, France). Our previously published transcriptomic data of 13 BPDCN (GSE89565) were used as a training cohort and a second cohort (n=37) was used as a validation to determine two subgroups of BPDCN, using bulk RNAsequencing (NEBNext Ultra II, New England Biolabs, Ipswich, MA, on NextSeq500, Illumina, San Diego, CA) on sorted populations (lymphocytes and blastic pDCs). Data were compared to 6 pDC and 4 lymphocyte populations obtained from healthy donors (HD) from French Blood Establishment. Paired MethylSequencing (MethylSeq) was performed on 30 blastic pDCs with SureSelectXT (Agilent, Santa Clara, CA) on NextSeq500 (Illumina). In-house pipelines on R were developed for transcription factor activities (DoRothEA/VIPER package), fusion transcript (Arriba/FusionCatcher/Starfusion), mutations (GATK), alternative splicing (Whippet), deconvolution (BisqueRNA), T-cell clone diversity (MIXCR), inference between blastic pDCs and lymphocytes (BulkSignalR), methylation status (Bismark/Methylkit/DMRichR and clusterProfiler). Probability of overall survival (OS) was determined using the Kaplan-Meyer method and log-rank test. Results: Two clusters were identified in the training cohort (median age= 61y[15-82], 11 males/13), one cluster with only men exhibiting MYB/MYC rearrangements (n=6) and another with apparent shorter OS, however without significance (n=7). These two clusters were not entirely transposed in the validation cohort (median age=67y[8-93], 26 males/37). As expected, epigenetic/splicing factor genes were highly mutated and MYB fusions were detected. Two clusters may be defined on blast fraction, but they were not strongly distinguished by unsupervised clustering. Interestingly, the lymphocyte fraction signature was shown to reflect the blasts. Combining z-scores of the 2 fractions, we note a correlation with two extreme cluster E1 and E2 without two distinct groups, but a continuity. E2 highly expressed pDC markers, HLA-II, IFN-I signalling, DNA replication, UV damages and fatty acid β-oxidation pathways. Deconvolution showed a various microenvironment, with higher T-cell clone diversity (unique TCRA/B). In contrast, E1 showed cell-cell signalling, NF-kB pathway, neural, hematopoiesis and TLR4 signatures in blasts, associated with T-cell activation, Th2 profile, IL4/IL13 signalling and ILC2 signatures,. Moreover, exon skipping was enhanced in contrast to E2 and HD pDCs. The inference study between blasts and lymphocytes showed interactions involving IL18, EGFR, IL13 in E2 and CD14, FGF in E1. Methylseq confirmed an involvement of neural and Wnt pathways in E1, while E2 showed hypomethylation of cellular response and immunological synapses targets. At last, despite short survival, our group 1 exhibited an adverse prognosis (OS 4.3 months vs 9.4 months, p=0.0431). Conclusions: even if BPDCN can be divided in two subgroups, this strategy splits intermediate patients who are very similar. We prefer to define a continuity between 2 extreme clusters. Despite the lack of a clear-cut distinction, the clusters harbour specific biological features and clinical impact. E1 confirm the C2 cluster of Künster et al., 2024 on an independent cohort. Our E2 cluster would be associated with UV damage, replication activation and fatty acid metabolism while E1 exhibit less differentiated signature, with NF-κB activation, neural signatures and different ability to chat with lymphocytes compared to E2. Prognosis and therapeutic impacts are hypothesized, NF-κB being targetable by proteasome inhibitors for instance.

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Hooper, J. E., M. Pérez-Alonso, J. R. Bermingham, M. Prout, B. A. Rocklein, M. Wagenbach, J. E. Edstrom, R. de Frutos, and M. P. Scott. "Comparative studies of Drosophila Antennapedia genes." Genetics 132, no. 2 (October 1, 1992): 453–69. http://dx.doi.org/10.1093/genetics/132.2.453.

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Abstract The Antennapedia (Antp) homeotic gene of Drosophila melanogaster controls cell fates and pattern formation in the epidermis, nervous system and mesoderm of thoracic segments. Its expression is controlled at the levels of transcription, alternative RNA splicing, polyadenylation and translation. Two nested Antp transcription units extend over 103 kb and produce sixteen different transcripts. We have compared the Antp genes of Drosophila virilis, Drosophila subobscura and D. melanogaster to determine which structural features are conserved and therefore may be important to the gene's function. The overall gene structures are similar. There are many conserved sequence blocks throughout the large introns, at least 15 kb upstream of the first promoter, and at least 3 kb downstream of the last polyadenylation site. Intron and exon sequence conservation around alternative splice sites indicates that alternative protein coding forms may also be conserved. Protein coding potential is perfectly conserved around the C-terminal homeodomain, well conserved in the N-terminal region, and more variable in the middle. The large size of the Antp gene may reflect a large number of control elements necessary for appropriate Antp protein expression. The conservation of transcript complexity suggests functional requirements for the different protein forms.

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Hou, Yue, Huan Huang, Wenqiao Hu, Hongde Liu, and Xiao Sun. "Histone modifications influence skipped exons inclusion." Journal of Bioinformatics and Computational Biology 15, no. 01 (February 2017): 1750003. http://dx.doi.org/10.1142/s0219720017500032.

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Alternative splicing (AS), by which individual genes can produce multiple mRNA, associates with genomic complexity, disease, and development. Histone modifications show important roles in both transcription initiation and mRNA splicing. Here, we intended to find the link between AS and histone modifications in flanking regions through analyzing publicly available data in two human cell lines, GM12878 and K562 cell lines. According to exon inclusion levels, exons were classified into three types, included skipped exons, excluded skipped exons and expressed constitutive exons. We revealed that the inclusion levels of skipped exons (SEs) were negatively correlated with the enrichment of active histone marks in SEs, indicating a role of histone modifications in AS. We also found that active histone modifications were enriched in the upstream exons of SEs, especially around 5[Formula: see text] splicing sites. We inferred that the histone modifications around the 5[Formula: see text] splicing sites in upstream exon of the SEs could help RNA Polymerase II complex to recruit the effector proteins and facilitate AS. It was indicated that nucleosome occupancy had little influence on the inclusion levels of SEs. At last, we proposed an integrated model that describe how histone modifications affected the pre-mRNA splicing.

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Bishop, David F., Xiaoye Schneider-Yin, Sonia Clavero, Han-Wook Yoo, Elisabeth I. Minder, and Robert J. Desnick. "Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively spliced transcripts." Blood 115, no. 5 (February 4, 2010): 1062–69. http://dx.doi.org/10.1182/blood-2009-04-218016.

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Abstract Splicing mutations account for approximately 10% of lesions causing genetic diseases, but few branchpoint sequence (BPS) lesions have been reported. In 3 families with autosomal recessive congenital erythropoietic porphyria (CEP) resulting from uroporphyrinogen III synthase (URO-synthase) deficiency, sequencing the promoter, all 10 exons and the intron/exon boundaries did not detect a mutation. Northern analyses of lymphoblast mRNAs from 2 patients and reverse-transcribed polymerase chain reaction (RT-PCR) of lymphoblast mRNAs from all 3 patients revealed multiple longer transcripts involving intron 9 and low levels of wild-type message. Sequencing intron 9 RT-PCR products and genomic DNA in each case revealed homozygosity for a novel BPS mutation (c.661-31T→G) and alternatively spliced transcripts containing 81, 246, 358, and 523 nucleotides from intron 9. RT-PCR revealed aberrant transcripts in both wild-type and CEP lymphoblasts, whereas BPS mutation reduced the wild-type transcript and enzyme activity in CEP lymphoblasts to approximately 10% and 15% of normal, respectively. Although the +81-nucleotide alternative transcript was in-frame, it only contributed approximately 0.2% of the lymphoblast URO-synthase activity. Thus, the BPS mutation markedly reduced the wild-type transcript and enzyme activity, thereby causing the disease. This is the first BPS mutation in the last intron, presumably accounting for the observed 100% intron retention without exon skipping.

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Chandradas, Sajiv, Jonathan G. Tardos, Ke Jiang, and Vladimir Bogdanov. "Post-Transcriptional Regulation of Tissue Factor Expression in Human Monocytic Cells: Identification of Novel Exonic Splicing Enhancers for the Spliceosomal Protein SRp40 and Intronic Elements Critical for Exon 5 Definition." Blood 112, no. 11 (November 16, 2008): 1031. http://dx.doi.org/10.1182/blood.v112.11.1031.1031.

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Abstract Background. Circulating monocytes play a critical role in the pathophysiology of many thrombotic disorders. These cells are known to exhibit complex post-transcriptional regulation of Tissue Factor (TF), the principal trigger of coagulation, which they express in two forms – full length TF(flTF), a highly thrombogenic integral membrane protein, and alternatively spliced TF (asTF), a secreted hypomorphic TF form. Biosynthesis of these two forms is achieved by inclusion or, alternatively, exclusion of TF exon 5 during pre-mRNA processing. Little is known about molecular mechanisms controlling alternative pre-mRNA splicing in human monocytes. We recently developed a mini-gene splicing reporter system pGL-hTF to evaluate the dynamics of exon 5 splicing, and determined that SR proteins ASF/SF2 and SRp55, which are abundantly expressed in human monocytes, take part in TF pre-mRNA processing by promoting exon 5 definition. Objective. To expand our exploration of cis-acting elements and spliceosomal proteins governing regulated biosynthesis of human TF in monocytic cells. Results. In silico analysis of exon 5 revealed six putative binding motifs termed exonic splicing enhancers (ESE) for SRp40, an SR protein whose expression pattern in human tissues is largely unknown. SRp40 mRNA and protein were detected in monocytic cell lines THP-1 and SC, as well as freshly isolated peripheral blood mononuclear cells (PBMC). Notably, the apparent molecular weight of the SRp40 protein in PBMC was substantially larger than that observed in monocytic cell lines, indicating that SRp40 in PBMC is likely to be hyperphosphorylated and may thus exhibit increased activity. To assess functionality of the putative SRp40 ESE in TF exon 5, base substitutions were designed to weaken each ESE and introduced into pGL-hTF by site directed mutagenesis. Resultant mutants were expressed in THP-1 cells, and the expression patterns analyzed by RT-PCR. Weakening of the ESE at position 44 potentiated inclusion of exon 5, while weakening of the ESE at position 86 resulted in increased exon 5 skipping. This suggests that SRp40 plays a complex, positional role in exon 5 definition. To further confirm functionality of the identified SRp40 ESE, combinatorial mutants were generated featuring weakened ESE for the SR proteins SRp55 (position 39, in close proximity to SRp40 ESE 44) and ASF/SF2 (positions 87–117): inactivation of these ESE was shown to promote skipping of exon 5. As expected, weakened SRp40 ESE 86, when combined with weakened SRp55 ESE, resulted in more severe skipping of exon 5. In contrast to that, weakened SRp40 ESE 44, when combined with weakened SRp55 ESE, potently restored inclusion of exon 5, whereas combining weakened SRp40 ESE 44 with weakened ASF/SF2 ESE yielded only a partial restoration of exon 5 inclusion. This indicates that SRp40 ESE 44 uniquely promotes production of the TF mRNA species encoding asTF, whereas SRp40 ESE 86 appears to behave similarly to classic ESE for spliceosomal proteins SRp55 and ASF/SF2. Using pGL-hTF, we also evaluated major regulatory elements of intron 4: this relatively short intron precedes the variable exon 5 and is thus likely to be critical for its spliceosomal definition. To identify a branchpoint (BP) adenosine (A), we replaced each A within the last 100 bp of the intron with a non-canonical BP base (G); surprisingly, these mutations did not affect the degree of exon 5 inclusion, indicating that intron 4 features a highly unusual BP motif. In contrast to that, mutations within the polypyrimidine tract (PT) of intron 4 revealed that the entire PT sequence is critical to exon 5 definition: compared to wild type pGL-hTF, a pGL-hTF mutant featuring extensive purine substitutions within the PT generated ~11.4 times fewer exon 5 containing amplicons (p=0.0001), as detected by our sensitive molecular beacon qPCR assay. Conclusions. We show for the first time that the spliceosomal protein SRp40 is expressed in human monocytes and participates in TF biosynthesis. Using our reporter system, we identified two functional ESE for SRp40 in the variable exon 5. SRp40 appears to play a complex, positional role in production of TF protein variants with a distinct coagulant potential. Furthering our knowledge about the post-transcriptional mechanisms of TF biosynthesis in circulating monocytes is very likely to facilitate development of qualitatively novel classes of anti-thrombotic therapeutic agents.

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Laudadio, Jennifer, Michael W. N. Deininger, Michael J. Mauro, Brian J. Druker, and Richard D. Press. "An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Three CML Patients Undergoing Kinase Inhibitor Therapy." Blood 110, no. 11 (November 16, 2007): 1953. http://dx.doi.org/10.1182/blood.v110.11.1953.1953.

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Abstract Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia, a minority acquire mutations in the kinase domain (KD) that cause imatinib resistance. The spectrum of KD mutations thus far discovered, although quite heterogeneous, includes almost exclusively single nucleotide substitutions in key amino acids regulating drug binding or BCR-ABL function. Here, we describe a KD insertion/truncation mutation in 3 CML patients undergoing kinase inhibitor therapy. Two of these patients were being treated with imatinib (for 12 and 17 months), and one with dasatinib (for 13 months after a prior relapse while on imatinib). Suspected drug resistance was assessed by direct DNA sequencing of a BCR-ABL PCR product extending to the end of the kinase domain. Each of these 3 patients had 35 nucleotides from ABL intron 8 inserted at the normal exon 8–9 splice junction, after nucleotide 1423 (amino acid 475) of Genbank cDNA clone NM_005157. In all 3 cases, the mutation was co-expressed with wild type BCR-ABL sequence. The inserted sequence is derived from intron 8, beginning 1151 bp downstream from the normal splice donor site at the end of exon 8. This 35 bp intronic sequence is flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion creates a premature translational stop codon after 10 intron-encoded amino acids (figure), thus truncating 653 C-terminal amino acids including part of the KD and the entire last exon region - including a proline-rich domain, 3 nuclear localization signals, a DNA-binding domain, an actin-binding domain, and a nuclear export signal. These 3 insertion mutation cases were detected in our diagnostic clinical molecular pathology laboratory after sequencing 174 cases referred to us for suspected kinase inhibitor resistance, 78 of which contained a detectable mutation. The estimated prevalence of the exon 8/9 insertion/truncation mutation is then approximately 1.7% among patients with suspected drug resistance, and this mutation constitutes approximately 3.8% of all mutations. Conclusion: Kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy. The functional significance in terms of kinase activity and drug resistance remains to be addressed. Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157). Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157).

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DODE, Leonard, Frank WUYTACK, Patrick F. J. KOOLS, Fouzia BABA-AISSA, Luc RAEYMAEKERS, Filip BRIK, Wim J. M. VAN DE VEN, and Rik CASTEELS. "cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene." Biochemical Journal 318, no. 2 (September 1, 1996): 689–99. http://dx.doi.org/10.1042/bj3180689.

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cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5´ region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3´-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5´-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177–6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

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Post, T. W., M. A. Arce, M. K. Liszewski, E. S. Thompson, J. P. Atkinson, and D. M. Lublin. "Structure of the gene for human complement protein decay accelerating factor." Journal of Immunology 144, no. 2 (January 15, 1990): 740–44. http://dx.doi.org/10.4049/jimmunol.144.2.740.

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Abstract Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32. These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR). A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones. The human DAF gene spans approximately 40 kb and consists of 11 exons. The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb. SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence. This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H. Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons. Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing. The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region. The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb. An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family. The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.

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Van de Wetering, M., J. Castrop, V. Korinek, and H. Clevers. "Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties." Molecular and Cellular Biology 16, no. 3 (March 1996): 745–52. http://dx.doi.org/10.1128/mcb.16.3.745.

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Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer.

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Risitano, Antonio M., Elisa Seneca, Ludovica Marando, Bianca Serio, Carmine Selleri, Giulia Scalia, Luigi Del Vecchio, et al. "Subcutaneous Alemtuzumab Is a Safe and Effective Treatment for Global or Single-Lineage Immune-Mediated Marrow Failures: a Survey from the EBMT-WPSAA." Blood 112, no. 11 (November 16, 2008): 1042. http://dx.doi.org/10.1182/blood.v112.11.1042.1042.

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Abstract Acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in aplastic anemia (AA), or may selectively involve single lineages, as in pure red cell (PRCA) or in pure white cell aplasias (PWCA). Because of their common cellular immune-mediated pathophysiology, standard treatment for these conditions includes immunosuppression (IS), which may differ according to the specific disease. We investigated an experimental IS regimen based on the anti-CD52 antibody alemtuzumab (MabCampath®, ALE); the study included a phase II/III prospective trial, as well as a collection of retrospective cases. A total of 32 patients have been treated by ALE (18 SAA, 10 PRCA and 4 PWCA), fourteen of them (mostly PRCA) having not received previous IS. The most utilized schedule (as defined in the prospective trial) was 3,10,30,30,30 mg (total dose 103), administered subcutaneously in consecutive days, with adequate premedication; the last dose was amended in PRCA and PWCA patients (total dose 73 mg). In the prospective trial, all patients also received oral low dose cyclosporine A (1 mg/kg) from day 7, and an intensive anti-infectious prophylaxis, which included oral valgancyclovir and cotrimoxazol. All patients completed the treatment with unrelevant injection-related side effect (fever and/or rash in some cases) and absence of laboratory abnormalities. Complete lympho-ablation was observed in all patients within 2–3 days, which persisted for several weeks; transient worsening of neutropenia and/or thrombocytopenia were observed in some cases. The median follow up was 12 months; there were 5 deaths, only one was possibly related to the treatment. In the prospective trial (n=23), infectious events were rare: a single FUO, associated with fatal complication of an underlying atrial fibrillation, other four viral infections (1 VZV with shingles, 2 HSV and 1 flu), all resolving quickly. No CMV or EBV disease was observed, even if 3 border-line CMV reactivations were documented (after discontinuation of the antiviral prophylaxis), promptly resolved by preemptive valganciclovir. One HBV reactivation without hepatitis required lamivudine. The response rate was globally 61% (42% CR and 19% PR), which raised to 73% (50% CR and 23% PR) when only patients with a follow up of at least 4 months were considered. In the more homogeneous cohort of the prospective trial, response rate was analyzed according to the underlying disease. Among 10 AA treated (5 as first line), 7 had an adequate follow up and showed 4 CR (57%) and 2 PR (29%). Response rate was even higher in 10 PRCA (8 as first line): 7 were evaluable for response, with 5 CR (71%) and 1 PR (14%); the 1 non responding patient subsequently showed evolution to MDS. Finally, 2 of 3 PWCA achieved a CR (66%), with the remaining showing early progression to MDS. Among the responding patients, relapses were quite frequent, even while on cyclosporine: 3/6 SAAs, 5/6 PRCAs and 1/2 PWCA. Relapses were successfully treated by additional ALE (as single shoots or complete courses). Immune reconstitution was delayed up to several months, especially affecting the CD4+ compartment; this was also due to additional ALE needed to treat or to prevent relapses. In conclusion, subcutaneous ALE is a feasible and safe IS regimen for patients suffering from immune-mediated marrow failure syndromes. Preliminary results suggest excellent efficacy, even if responses may be quite late (3–4 months); relapses often occur, but can be easily managed by ALE retreatment. ALE is an excellent alternative to standard IS regimen, and deserves systematic investigation in bone marrow failure patients.

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Maggi, Jordi, James V. M. Hanson, Lisa Kurmann, Samuel Koller, Silke Feil, Christina Gerth-Kahlert, and Wolfgang Berger. "Retinal Dystrophy Associated with Homozygous Variants in NRL." Genes 15, no. 12 (December 12, 2024): 1594. https://doi.org/10.3390/genes15121594.

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Background/Objectives: Neural retina leucine zipper (NRL) is a transcription factor involved in the differentiation of rod photoreceptors. Pathogenic variants in the gene encoding NRL have been associated with autosomal dominant retinitis pigmentosa and autosomal recessive clumped pigmentary retinal degeneration. Only a dozen unrelated families affected by recessive NRL-related retinal dystrophy have been described. The purpose of this study was to expand the genotypic spectrum of this disease by reporting clinical and genetic findings of two unrelated families. Methods: Index patients affected by retinal dystrophy were genetically tested by whole-exome sequencing (WES) and whole-genome sequencing (WGS). Segregation analysis within the families was performed for candidate variants. A minigene assay was performed to functionally characterize a variant suspected to affect splicing. Results: Variant filtering revealed homozygous NRL variants in both families. The variant in patient A was a small deletion encompassing the donor splice site of exon 1 of transcript NM_006177.3. The minigene assay revealed that this variant led to two aberrant transcripts that used alternative cryptic donor splice sites located in intron 1. In patient B, a stop-gain variant was identified in the last exon of NRL in a homozygous state due to maternal uniparental disomy of chromosome 14. Conclusions: Our study expands the genotypic spectrum of autosomal recessive NRL-related retinal dystrophy. Moreover, it underscores the importance of actively maintaining bioinformatic pipelines for variant detection and the utility of minigene assays in functionally characterizing candidate splicing variants.

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Wang, Hui-Qin, Tian He, Xiao-Feng Yu, and Ya-Nan Huo. "A novel pathogenic splicing mutation of RPGR in a Chinese family with X-linked retinitis pigmentosa verified by minigene splicing assay." International Journal of Ophthalmology 16, no. 10 (October 18, 2023): 1595–600. http://dx.doi.org/10.18240/ijo.2023.10.06.

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AIM: To report a novel splicing mutation in the RPGR gene (encoding retinitis pigmentosa GTPase regulator) in a three-generation Chinese family with X-linked retinitis pigmentosa (XLRP). METHODS: Comprehensive ophthalmic examinations including best corrected visual acuity, fundus photography, vision field, and pattern-visual evoked potential were performed to identify the disease phenotype of a six-year-old boy from the family (proband). Genomic DNA was extracted from peripheral blood of five available members of the pedigree. Whole-exome sequencing (WES), Sanger sequencing, and pSPL3-based exon trapping were used to investigate the aberrant splicing of RPGR. Human Splice Finder v3.1 and NNSPLICE v0.9 were used for in silico prediction of splice site variants. RESULTS: The proband was diagnosed as having retinitis pigmentosa (RP). He had severe symptoms with early onset. A novel splicing mutation, c.619+1G>C in RPGR was identified in the proband by WES and in four family members by Sanger sequencing. Minigene splicing assays verified that c.619+1G>C in RPGR would result in the formation of a damaging alternative transcript in which the last 91 bp of exon 6 were skipped, leading to the subsequent deletion of 623 correct amino acids (c.529_619del p.Val177Glnfs*16). CONCLUSION: We identify a novel splice donor site mutation causing aberrant splicing of RPGR. Our findings add to the catalog of pathological mutations of RPGR and further emphasize the functional importance of RPGR in RP pathogenesis and its complex clinical phenotypes.

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Sag, Erdal, Fuat Akal, Erdal Atalay, Ummusen Kaya Akca, Selcan Demir, Dilara Demirel, Ezgi Deniz Batu, Yelda Bilginer, and Seza Ozen. "Anti-IL1 treatment in colchicine-resistant paediatric FMF patients: real life data from the HELIOS registry." Rheumatology 59, no. 11 (April 19, 2020): 3324–29. http://dx.doi.org/10.1093/rheumatology/keaa121.

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Abstract Objectives FMF is a prototype of autoinflammatory diseases associated with excess IL1 production. Anti-IL1 treatments are the first-line alternatives in colchicine-resistant/intolerant FMF patients. We aimed to investigate the efficacy and safety of anti-IL1 treatment in paediatric FMF patients in our local [Hacettepe univErsity eLectronIc research fOrmS (HELIOS)] registry. Methods HELIOS is a web-based biologic drug registry for paediatric rheumatology patients. We have analysed the clinical features, disease activity parameters, treatment responses and safety outcomes in FMF patients treated with anti-IL1 agents. Results Forty paediatric FMF patients (34 continuous and six on-demand use) were included. Among the continuously treated group (61.7% female), the mean age at the start of colchicine was 5.55 (3.87) years. Age at onset of the anti-IL1 treatment was 11.47 (5.41) years with a mean follow-up duration of 3.87 (1.96) years. Apart from two, all patients had biallelic exon-10 mutations. We also gave anti-IL1 treatment on an on-demand basis in six patients. Anakinra was used as the first-line anti-IL1 treatment. During the last visit, six patients were treated with anakinra and 28 patients with canakinumab. Anti-IL1 treatment decreased the CRP levels and number and severity of the attacks. There were three hospitalizations reported due to mild infections. Eleven patients had local skin reactions, two patients had leucopenia with anakinra and one patient had thrombocytopenia with canakinumab. There was no malignancy or other severe adverse reactions. Conclusion Anakinra and canakinumab are efficient and safe alternatives in colchicine-resistant or -intolerant paediatric FMF patients. We also, for the first time, report on-demand use of anti-IL1 in paediatric FMF patients.

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Journal articles on the topic 'Alternative last exon (ALE)'

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