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MacDonald, C. C., J. Wilusz, and T. Shenk. "The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location." Molecular and Cellular Biology 14, no. 10 (October 1994): 6647–54. http://dx.doi.org/10.1128/mcb.14.10.6647-6654.1994.
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The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.
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MacDonald, C. C., J. Wilusz, and T. Shenk. "The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location." Molecular and Cellular Biology 14, no. 10 (October 1994): 6647–54. http://dx.doi.org/10.1128/mcb.14.10.6647.
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The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.
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Mangus, David A., Mandy M. Smith, Jennifer M. McSweeney, and Allan Jacobson. "Identification of Factors Regulating Poly(A) Tail Synthesis and Maturation." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4196–206. http://dx.doi.org/10.1128/mcb.24.10.4196-4206.2004.
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ABSTRACT Posttranscriptional maturation of the 3′ end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3′-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1Δ and pbp1Δ cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).
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Chen, J., and C. Moore. "Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA." Molecular and Cellular Biology 12, no. 8 (August 1992): 3470–81. http://dx.doi.org/10.1128/mcb.12.8.3470-3481.1992.
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Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.
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Chen, J., and C. Moore. "Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA." Molecular and Cellular Biology 12, no. 8 (August 1992): 3470–81. http://dx.doi.org/10.1128/mcb.12.8.3470.
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Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.
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Zhao, Jing, Linda Hyman, and Claire Moore. "Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis." Microbiology and Molecular Biology Reviews 63, no. 2 (June 1, 1999): 405–45. http://dx.doi.org/10.1128/mmbr.63.2.405-445.1999.
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SUMMARY Formation of mRNA 3′ ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3′ ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.
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Ryner, L. C., Y. Takagaki, and J. L. Manley. "Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly." Molecular and Cellular Biology 9, no. 4 (April 1989): 1759–71. http://dx.doi.org/10.1128/mcb.9.4.1759-1771.1989.
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To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.
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Ryner, L. C., Y. Takagaki, and J. L. Manley. "Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly." Molecular and Cellular Biology 9, no. 4 (April 1989): 1759–71. http://dx.doi.org/10.1128/mcb.9.4.1759.
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To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.
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Morlando, Mariangela, Paolo Greco, Bernhard Dichtl, Alessandro Fatica, Walter Keller, and Irene Bozzoni. "Functional Analysis of Yeast snoRNA and snRNA 3′-End Formation Mediated by Uncoupling of Cleavage and Polyadenylation." Molecular and Cellular Biology 22, no. 5 (March 1, 2002): 1379–89. http://dx.doi.org/10.1128/mcb.22.5.1379-1389.2002.
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ABSTRACT Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.
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McCracken, Susan, Mark Lambermon, and Benjamin J. Blencowe. "SRm160 Splicing Coactivator Promotes Transcript 3′-End Cleavage." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 148–60. http://dx.doi.org/10.1128/mcb.22.1.148-160.2002.
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ABSTRACT Individual steps in the processing of pre-mRNA, including 5′-end cap formation, splicing, and 3′-end processing (cleavage and polyadenylation) are highly integrated and can influence one another. In addition, prior splicing can influence downstream steps in gene expression, including export of mRNA from the nucleus. However, the factors and mechanisms coordinating these steps in the maturation of pre-mRNA transcripts are not well understood. In the present study we demonstrate that SRm160 (for serine/arginine repeat-related nuclear matrix protein of 160 kDa), a coactivator of constitutive and exon enhancer-dependent splicing, participates in 3′-end formation. Increased levels of SRm160 promoted the 3′-end cleavage of transcripts both in vivo and in vitro. Remarkably, at high levels in vivo SRm160 activated the 3′-end cleavage and cytoplasmic accumulation of unspliced pre-mRNAs, thereby uncoupling the requirement for splicing to promote the 3′-end formation and nuclear release of these transcripts. Consistent with a role in 3′-end formation coupled to splicing, SRm160 was found to associate specifically with the cleavage polyadenylation specificity factor and to stimulate the 3′-end cleavage of splicing-active pre-mRNAs more efficiently than that of splicing-inactive pre-mRNAs in vitro. The results provide evidence for a role for SRm160 in mRNA 3′-end formation and suggest that the level of this splicing coactivator is important for the proper coordination of pre-mRNA processing events.
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Fang, Liying, Lina Guo, Min Zhang, Xianchun Li, and Zhongyuan Deng. "Analysis of Polyadenylation Signal Usage with Full-Length Transcriptome in Spodoptera frugiperda (Lepidoptera: Noctuidae)." Insects 13, no. 9 (September 2, 2022): 803. http://dx.doi.org/10.3390/insects13090803.
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During the messenger RNA (mRNA) maturation process, RNA polyadenylation is a key step, and is coupled to the termination of transcription. Various cis-acting elements near the cleavage site and their binding factors would affect the process of polyadenylation, and AAUAAA, a highly conserved hexamer, was the most important polyadenylation signal (PAS). PAS usage is one of the critical modification determinants targeted at mRNA post-transcription. The full-length transcriptome has recently generated a massive amount of sequencing data, revealing poly(A) variation and alternative polyadenylation (APA) in Spodoptera frugiperda. We identified 50,616 polyadenylation signals in Spodoptera frugiperda via analysis of full-length transcriptome combined with expression Sequence Tags Technology (EST). The polyadenylation signal usage in Spodoptera frugiperda is conserved, and it is similar to that of flies and other animals. AAUAAA and AUUAAA are the most highly conserved polyadenylation signals of all polyadenylation signals we identified. Additionally, we found the U/GU-rich downstream sequence element (DSE) in the cleavage site. These results demonstrate that APA in Spodoptera frugiperda plays a significant role in root growth and development. This is the first polyadenylation signal usage analysis in agricultural pests, which can deepen our understanding of Spodoptera frugiperda and provide a theoretical basis for pest control.
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Qu, Xiangping, Søren Lykke-Andersen, Tommy Nasser, Cyril Saguez, Edouard Bertrand, Torben Heick Jensen, and Claire Moore. "Assembly of an Export-Competent mRNP Is Needed for Efficient Release of the 3′-End Processing Complex after Polyadenylation." Molecular and Cellular Biology 29, no. 19 (July 27, 2009): 5327–38. http://dx.doi.org/10.1128/mcb.00468-09.
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ABSTRACT Before polyadenylated mRNA is exported from the nucleus, the 3′-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A)+ RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3′-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3′-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3′-end processing complex once polyadenylation is complete.
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Steinmetz, Eric J., and David A. Brow. "Ssu72 Protein Mediates Both Poly(A)-Coupled and Poly(A)-Independent Termination of RNA Polymerase II Transcription." Molecular and Cellular Biology 23, no. 18 (September 15, 2003): 6339–49. http://dx.doi.org/10.1128/mcb.23.18.6339-6349.2003.
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ABSTRACT Termination of transcription by RNA polymerase II (Pol II) is a poorly understood yet essential step in eukaryotic gene expression. Termination of pre-mRNA synthesis is coupled to recognition of RNA signals that direct cleavage and polyadenylation of the nascent transcript. Termination of nonpolyadenylated transcripts made by Pol II in the yeast Saccharomyces cerevisiae, including the small nuclear and small nucleolar RNAs, requires distinct RNA elements recognized by the Nrd1 protein and other factors. We have used genetic selection to characterize the terminator of the SNR13 snoRNA gene, revealing a bipartite structure consisting of an upstream element closely matching a Nrd1-binding sequence and a downstream element similar to a cleavage/polyadenylation signal. Genome-wide selection for factors influencing recogniton of the SNR13 terminator yielded mutations in the gene coding for the essential Pol II-binding protein Ssu72. Ssu72 has recently been found to associate with the pre-mRNA cleavage/polyadenylation machinery, and we find that an ssu72 mutation that disrupts Nrd1-dependent termination also results in deficient poly(A)-dependent termination. These findings extend the parallels between the two termination pathways and suggest that they share a common mechanism to signal Pol II termination.
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Lepennetier, Gildas, and Francesco Catania. "Exploring the Impact of Cleavage and Polyadenylation Factors on Pre-mRNA Splicing Across Eukaryotes." G3 Genes|Genomes|Genetics 7, no. 7 (July 1, 2017): 2107–14. http://dx.doi.org/10.1534/g3.117.041483.
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Abstract In human, mouse, and Drosophila, the spliceosomal complex U1 snRNP (U1) protects transcripts from premature cleavage and polyadenylation at proximal intronic polyadenylation signals (PAS). These U1-mediated effects preserve transcription integrity, and are known as telescripting. The watchtower role of U1 throughout transcription is clear. What is less clear is whether cleavage and polyadenylation factors (CPFs) are simply patrolled or if they might actively antagonize U1 recruitment. In addressing this question, we found that, in the introns of human, mouse, and Drosophila, and of 14 other eukaryotes, including multi- and single-celled species, the conserved AATAAA PAS—a major target for CPFs—is selected against. This selective pressure, approximated using DNA strand asymmetry, is detected for peripheral and internal introns alike. Surprisingly, it is more pronounced within—rather than outside—the action range of telescripting, and particularly intense in the vicinity of weak 5′ splice sites. Our study uncovers a novel feature of eukaryotic genes: that the AATAAA PAS is universally counter-selected in spliceosomal introns. This pattern implies that CPFs may attempt to access introns at any time during transcription. However, natural selection operates to minimize this access. By corroborating and extending previous work, our study further indicates that CPF access to intronic PASs might perturb the recruitment of U1 to the adjacent 5′ splice sites. These results open the possibility that CPFs may impact the splicing process across eukaryotes.
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Takagaki, Yoshio, and James L. Manley. "Complex Protein Interactions within the Human Polyadenylation Machinery Identify a Novel Component." Molecular and Cellular Biology 20, no. 5 (March 1, 2000): 1515–25. http://dx.doi.org/10.1128/mcb.20.5.1515-1525.2000.
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ABSTRACT Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.
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Peterson, M. L., M. B. Bryman, M. Peiter, and C. Cowan. "Exon size affects competition between splicing and cleavage-polyadenylation in the immunoglobulin mu gene." Molecular and Cellular Biology 14, no. 1 (January 1994): 77–86. http://dx.doi.org/10.1128/mcb.14.1.77-86.1994.
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The alternative RNA processing of microseconds and microns mRNAs from a single primary transcript depends on competition between a cleavage-polyadenylation reaction to produce microseconds mRNA and a splicing reaction to produce microns mRNA. The ratio of microseconds to microns mRNA is regulated during B-cell maturation; relatively more spliced microns mRNA is made in B cells than in plasma cells. The balance between the efficiencies of splicing and cleavage-polyadenylation is critical to the regulation. The mu gene can be modified to either reduce or improve the efficiency of each reaction and thus alter the ratio of the two RNAs produced. However, as long as neither reaction is so strong that it totally dominates, expression of the modified mu genes is regulated in B cells and plasma cells. The current experiments reveal a relationship between the C mu 4 exon size and the microseconds/microns expression ratio. The shorter the distance between the C mu 4 5' splice site and the nearest upstream 3' splice site, the more spliced microns mRNA was produced. Conversely, when this exon was expanded, more microseconds mRNA was produced. Expression from these mu genes with altered exon sizes were regulated between B cells and plasma cells. Since RNA processing in the mu gene can be considered a competition between defining the C mu 4 exon as an internal exon (in microns mRNA) versus a terminal exon (in microseconds mRNA), exon size may affect the competition among factors interacting with this exon.
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Peterson, M. L., M. B. Bryman, M. Peiter, and C. Cowan. "Exon size affects competition between splicing and cleavage-polyadenylation in the immunoglobulin mu gene." Molecular and Cellular Biology 14, no. 1 (January 1994): 77–86. http://dx.doi.org/10.1128/mcb.14.1.77.
Full textAbstract:
The alternative RNA processing of microseconds and microns mRNAs from a single primary transcript depends on competition between a cleavage-polyadenylation reaction to produce microseconds mRNA and a splicing reaction to produce microns mRNA. The ratio of microseconds to microns mRNA is regulated during B-cell maturation; relatively more spliced microns mRNA is made in B cells than in plasma cells. The balance between the efficiencies of splicing and cleavage-polyadenylation is critical to the regulation. The mu gene can be modified to either reduce or improve the efficiency of each reaction and thus alter the ratio of the two RNAs produced. However, as long as neither reaction is so strong that it totally dominates, expression of the modified mu genes is regulated in B cells and plasma cells. The current experiments reveal a relationship between the C mu 4 exon size and the microseconds/microns expression ratio. The shorter the distance between the C mu 4 5' splice site and the nearest upstream 3' splice site, the more spliced microns mRNA was produced. Conversely, when this exon was expanded, more microseconds mRNA was produced. Expression from these mu genes with altered exon sizes were regulated between B cells and plasma cells. Since RNA processing in the mu gene can be considered a competition between defining the C mu 4 exon as an internal exon (in microns mRNA) versus a terminal exon (in microseconds mRNA), exon size may affect the competition among factors interacting with this exon.
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Amrani, N., M. Minet, M. Le Gouar, F. Lacroute, and F. Wyers. "Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro." Molecular and Cellular Biology 17, no. 7 (July 1997): 3694–701. http://dx.doi.org/10.1128/mcb.17.7.3694.
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In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.
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Zhao, Xiaomin, Daniel Öberg, Margaret Rush, Joanna Fay, Helen Lambkin, and Stefan Schwartz. "A 57-Nucleotide Upstream Early Polyadenylation Element in Human Papillomavirus Type 16 Interacts with hFip1, CstF-64, hnRNP C1/C2, and Polypyrimidine Tract Binding Protein." Journal of Virology 79, no. 7 (April 1, 2005): 4270–88. http://dx.doi.org/10.1128/jvi.79.7.4270-4288.2005.
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ABSTRACT We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3′ UTR) in HPV-16 gene expression. We found that deletion of the early 3′ UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3′ half of the early 3′ UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3′ splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.
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Danckwardt, Sven, Marc Gentzel, Niels H. Gehring, Isabelle Kaufmann, Gabriele Neu-Yilik, Matthias Wilm, Matthias W. Hentze, and Andreas E. Kulozik. "The Physiology of Prothrombin Gene Expression Integrates RNA Polyadenylation and Splicing in a Novel Regulatable 3′ RNP-Complex." Blood 108, no. 11 (November 16, 2006): 1601. http://dx.doi.org/10.1182/blood.v108.11.1601.1601.
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Abstract The functional analysis of the common prothrombin (F2) 20210*A allele has recently revealed gain-of-function of 3′end processing as a novel genetic mechanism predisposing to human disease. The general susceptibility of the F2 mRNA for gain-of-function is further exemplified by F2 20209*T and F2 20221*T, and can be explained by an unusual architecture of non-canonical 3′end formation sequence elements: Specifically, the F2 3′ untranslated region (3′UTR) contains a stimulatory upstream sequence element (USE) that compensates for the weak functional activities of the cleavage site and the downstream U-rich element in the F2 3′ flanking sequence. We now show that the F2 USE promotes 3′end formation in a position- and sequence-dependent manner, stimulating the step of mRNA polyadenylation rather than cleavage, and identify specific proteins that interact with the USE. Unexpectedly, the USE RNP includes splicing factors, components of the 3′end processing machinery and AU-rich sequence element-binding proteins (ARE-BP). We demonstrate that the splicing factors U2AF35 and U2AF65, hnRNPI/PTB, PSF/SFPQ and p54nrb/NonO promote 3′end formation via the USE contained in the 3′UTR uncovering a novel and more general functional link between these splicing factors and mRNA 3′ end formation. We propose a model of USE-directed 3′ end processing that involves a novel mRNP that integrates different nuclear pre-mRNA processing steps. Furthermore, the involvement of ARE-BP in this mRNP reveals an intriguing potential for a post-transcriptional regulation of prothrombin gene expression through external stimuli. Our data thus implicate USE-dependent RNP-complex formation in the regulated physiology of prothrombin gene expression specifically and in hemostasis (and other thrombin-dependent processes) more generally.
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Fischer, Jonathan, Yun S. Song, Nir Yosef, Julia di Iulio, L. Stirling Churchman, and Mordechai Choder. "The yeast exoribonuclease Xrn1 and associated factors modulate RNA polymerase II processivity in 5' and 3' gene regions." Journal of Biological Chemistry 295, no. 33 (June 9, 2020): 11435–54. http://dx.doi.org/10.1074/jbc.ra120.013426.
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mRNA levels are determined by the balance between mRNA synthesis and decay. Protein factors that mediate both processes, including the 5'-3' exonuclease Xrn1, are responsible for a cross-talk between the two processes that buffers steady-state mRNA levels. However, the roles of these proteins in transcription remain elusive and controversial. Applying native elongating transcript sequencing (NET-seq) to yeast cells, we show that Xrn1 functions mainly as a transcriptional activator and that its disruption manifests as a reduction of RNA polymerase II (Pol II) occupancy downstream of transcription start sites. By combining our sequencing data and mathematical modeling of transcription, we found that Xrn1 modulates transcription initiation and elongation of its target genes. Furthermore, Pol II occupancy markedly increased near cleavage and polyadenylation sites in xrn1Δ cells, whereas its activity decreased, a characteristic feature of backtracked Pol II. We also provide indirect evidence that Xrn1 is involved in transcription termination downstream of polyadenylation sites. We noted that two additional decay factors, Dhh1 and Lsm1, seem to function similarly to Xrn1 in transcription, perhaps as a complex, and that the decay factors Ccr4 and Rpb4 also perturb transcription in other ways. Interestingly, the decay factors could differentiate between SAGA- and TFIID-dominated promoters. These two classes of genes responded differently to XRN1 deletion in mRNA synthesis and were differentially regulated by mRNA decay pathways, raising the possibility that one distinction between these two gene classes lies in the mechanisms that balance mRNA synthesis with mRNA decay.
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Yang, Jing, Ying Cao, and Ligeng Ma. "Co-Transcriptional RNA Processing in Plants: Exploring from the Perspective of Polyadenylation." International Journal of Molecular Sciences 22, no. 7 (March 24, 2021): 3300. http://dx.doi.org/10.3390/ijms22073300.
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Most protein-coding genes in eukaryotes possess at least two poly(A) sites, and alternative polyadenylation is considered a contributing factor to transcriptomic and proteomic diversity. Following transcription, a nascent RNA usually undergoes capping, splicing, cleavage, and polyadenylation, resulting in a mature messenger RNA (mRNA); however, increasing evidence suggests that transcription and RNA processing are coupled. Plants, which must produce rapid responses to environmental changes because of their limited mobility, exhibit such coupling. In this review, we summarize recent advances in our understanding of the coupling of transcription with RNA processing in plants, and we describe the possible spatial environment and important proteins involved. Moreover, we describe how liquid–liquid phase separation, mediated by the C-terminal domain of RNA polymerase II and RNA processing factors with intrinsically disordered regions, enables efficient co-transcriptional mRNA processing in plants.
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Yu, Simei, Antonio Jordán-Pla, Antoni Gañez-Zapater, Shruti Jain, Anna Rolicka, Ann-Kristin Östlund Farrants, and Neus Visa. "SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3′ end processing." Nucleic Acids Research 46, no. 16 (May 31, 2018): 8557–73. http://dx.doi.org/10.1093/nar/gky438.
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Hodgman, Rebecca, Joyce Tay, Raul Mendez, and Joel D. Richter. "CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes." Development 128, no. 14 (July 15, 2001): 2815–22. http://dx.doi.org/10.1242/dev.128.14.2815.
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In both vertebrates and invertebrates, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. In Xenopus oocytes, where most of the biochemical details of this process have been examined, polyadenylation is controlled by CPEB, a sequence-specific RNA binding protein. The activity of CPEB, which is to recruit cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase (PAP) into an active cytoplasmic polyadenylation complex, is controlled by Eg2-catalyzed phosphorylation. Soon after CPEB phosphorylation and resulting polyadenylation take place, the interaction between maskin, a CPEB-associated factor, and eIF4E, the cap-binding protein, is destroyed, which results in the recruitment of mRNA into polysomes. Polyadenylation also occurs in maturing mouse oocytes, although the biochemical events that govern the reaction in these cells are not known. In this study, we have examined the phosphorylation of CPEB and have assessed the necessity of this protein for polyadenylation in maturing mouse oocytes. Immunohistochemistry has revealed that all the factors that control polyadenylation and translation in Xenopus oocytes (CPEB, CPSF, PAP, maskin, and IAK1, the murine homologue of Eg2) are also present in the cytoplasm of mouse oocytes. After the induction of maturation, a kinase is activated that phosphorylates CPEB on a critical regulatory residue, an event that is essential for CPEB activity. A peptide that competitively inhibits the activity of IAK1/Eg2 blocks the progression of meiosis in injected oocytes. Finally, a CPEB protein that acts as a dominant negative mutation because it cannot be phosphorylated by IAK1/Eg2, prevents cytoplasmic polyadenylation. These data indicate that cytoplasmic polyadenylation in mouse oocytes is mediated by IAK1/Eg2-catalyzed phosphorylation of CPEB.
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Wigley, P. L., M. D. Sheets, D. A. Zarkower, M. E. Whitmer, and M. Wickens. "Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA." Molecular and Cellular Biology 10, no. 4 (April 1990): 1705–13. http://dx.doi.org/10.1128/mcb.10.4.1705-1713.1990.
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mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.
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Wigley, P. L., M. D. Sheets, D. A. Zarkower, M. E. Whitmer, and M. Wickens. "Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA." Molecular and Cellular Biology 10, no. 4 (April 1990): 1705–13. http://dx.doi.org/10.1128/mcb.10.4.1705.
Full textAbstract:
mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.
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Muniz, Lisa, Lee Davidson, and Steven West. "Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs." Molecular and Cellular Biology 35, no. 13 (April 20, 2015): 2218–30. http://dx.doi.org/10.1128/mcb.00123-15.
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Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3′-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.
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Zhao, Jing, Marco Kessler, Steffen Helmling, J. Patrick O’Connor, and Claire Moore. "Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor." Molecular and Cellular Biology 19, no. 11 (November 1, 1999): 7733–40. http://dx.doi.org/10.1128/mcb.19.11.7733.
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ABSTRACT CF II, a factor required for cleavage of the 3′ ends of mRNA precursor in Saccharomyces cerevisiae, has been shown to contain four polypeptides. The three largest subunits, Cft1/Yhh1, Cft2/Ydh1, and Brr5/Ysh1, are homologs of the three largest subunits of mammalian cleavage-polyadenylation specificity factor (CPSF), an activity needed for both cleavage and poly(A) addition. In this report, we show by protein sequencing and immunoreactivity that the fourth subunit of CF II is Pta1, an essential 90-kDa protein originally implicated in tRNA splicing. Yth1, the yeast homolog of the CPSF 30-kDa subunit, is not detected in this complex. Extracts prepared frompta1 mutant strains are impaired in the cleavage and the poly(A) addition of both GAL7 and CYC1substrates and exhibit little processing activity even after prolonged incubation. However, activity is efficiently rescued by the addition of purified CF II to the defective extracts. Extract from a strain with a mutation in the CF IA subunit Rna14 also restored processing, but extract from a brr5-1 strain did not. The amounts of Pta1 and other CF II subunits are reduced in pta1 strains, suggesting that levels of the subunits may be coordinately regulated. Coimmunoprecipitation experiments indicate that the CF II in extract can be found in a stable complex containing Pap1, CF II, and the Fip1 and Yth1 subunits of polyadenylation factor I. While purified CF II does not appear to retain the association with these other factors, this larger complex may be the form recruited onto pre-mRNA in vivo. The involvement of Pta1 in both steps of mRNA 3′-end formation supports the conclusion that CF II is the functional homolog of CPSF.
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Yang, M., Z. Fan, and I. A. Polejaeva. "1 Microinjection of CPE-Binding Protein Polyadenylated mRNA Increases Developmental Competence of Bovine Oocytes In Vitro." Reproduction, Fertility and Development 30, no. 1 (2018): 140. http://dx.doi.org/10.1071/rdv30n1ab1.
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Developmental competence is acquired during oocyte growth and maturation while oocytes undergo both nuclear and cytoplasmic changes. Completion of oocyte maturation and subsequent embryo development relies mostly on maternally synthesised and stored mRNAs at the transcriptionally quiescent phase. The temporal and spatial post-transcriptional and translational regulation of the stored mRNA in mammalian oocyte cytoplasm is essential for developmental competence of oocytes and is often controlled via cytoplasmic polyadenylation. Cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) is required for polyadenylation of most mRNAs during oocyte maturation. It has been reported that in vitro-matured oocytes with high developmental competence showed an increased level of CPEB mRNA in oocyte cytoplasm. Thus, we hypothesise that the introduction of exogenous CPEB mRNA into in vitro-matured oocytes could increase their developmental capability. In this study, we first synthesised polyadenylated CPEB mRNAs by in vitro transcription. Cumulus-oocyte complexes were recovered from slaughterhouse ovaries and subjected to in vitro maturation for 21 h. After the removal of cumulus cells, matured oocytes were parthenogenetically activated (5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL−1 cycloheximide). Each activated oocyte was injected with 5 to 10 pL of poly(A)-RNA solution (400 ng μL−1; CPEB mRNA and green fluorescent protein (GFP) mRNA for the injection group or GFP mRNA for the control group) using a micromanipulator. After injection, the oocytes were cultured in SOF medium supplemented with amino acids for 8 days. No difference was observed in cleavage rate between CPEB and control group. However, the blastocyst rate was significantly higher in the CPEB group than in the control (24.9 ± 2.9% v. 15.0 ± 4.5%; P < 0.05). Cleavage and blastocyst rates were analysed by one-way ANOVA. We also compared the gene expression profile of blastocysts derived from both groups. The blastocysts were collected individually and analysed by single-embryo RT-PCR. Twenty-two genes were selected for analysis based on their roles in genomic reprogramming and embryonic development and fell into 6 functional categories: growth regulatory factors, cell cycle regulation, imprinting, apoptosis, pluripotency and DNA methyltransferase. The single-embryo RT-PCR was performed using the Flex-Six integrated fluidic chip (Fluidigm Corp., South San Francsisco, CA, USA) on the BioMark platform (Fluidigm Corp.). Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. We found that 6 genes (H19, GRB10, DNMT1, CCNB1, CDK2, and SOX2) were up-regulated and 3 were down-regulated (DNMT2, BAX, and P53), along with the overexpression of CPEB gene (P < 0.05). Our results demonstrate that developmental competence can be improved by injecting exogenous CPEB mRNA into in vitro-matured metaphase II cattle oocytes, which reaffirms the essential role of CPEB in early embryonic development.
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Key, S. Catherine Silver, Tomokazu Yoshizaki, and Joseph S. Pagano. "The Epstein-Barr Virus (EBV) SM Protein Enhances Pre-mRNA Processing of the EBV DNA Polymerase Transcript." Journal of Virology 72, no. 11 (November 1, 1998): 8485–92. http://dx.doi.org/10.1128/jvi.72.11.8485-8492.1998.
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ABSTRACT The Epstein-Barr virus (EBV) DNA polymerase (pol) mRNA, which contains a noncanonical polyadenylation signal, UAUAAA, is cleaved and polyadenylated inefficiently (S. C. S. Key and J. S. Pagano, Virology 234:147–159, 1997). We postulated that the EBV early proteins SM and M, which appear to act posttranscriptionally and are homologs of herpes simplex virus (HSV) ICP27, might compensate for the inefficient processing ofpol pre-mRNA. Here we show that the SM and M proteins interact with each other in vitro. In addition, glutathioneS-transferase–SM/M fusion proteins precipitate the heterogeneous ribonucleoprotein (hnRNP) C1 splicing protein. Further, the SM protein is coimmunoprecipitated from SM-expressing cell extracts with an antibody to the hnRNP A1/A2 proteins, which are splicing and nuclear shuttling proteins. Finally, the amount of processed EBV DNA polymerase mRNA was increased three- to fourfold in a HeLa cell line expressing SM; this increase was not due to enhanced transcription. Thus, inefficient processing of EBV pol RNA by cellular cleavage and polyadenylation factors appears to be compensated for and may be regulated by the early EBV protein, SM, perhaps via RNA 3′-end formation.
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Ryner, L. C., Y. Takagaki, and J. L. Manley. "Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation." Molecular and Cellular Biology 9, no. 10 (October 1989): 4229–38. http://dx.doi.org/10.1128/mcb.9.10.4229-4238.1989.
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Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.
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Ryner, L. C., Y. Takagaki, and J. L. Manley. "Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation." Molecular and Cellular Biology 9, no. 10 (October 1989): 4229–38. http://dx.doi.org/10.1128/mcb.9.10.4229.
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Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.
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Bléoo, Stacey, Xuejun Sun, Michael J. Hendzel, John M. Rowe, Mary Packer, and Roseline Godbout. "Association of Human DEAD Box Protein DDX1 with a Cleavage Stimulation Factor Involved in 3′-End Processing of Pre-mRNA." Molecular Biology of the Cell 12, no. 10 (October 2001): 3046–59. http://dx.doi.org/10.1091/mbc.12.10.3046.
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DEAD box proteins are putative RNA helicases that function in all aspects of RNA metabolism, including translation, ribosome biogenesis, and pre-mRNA splicing. Because many processes involving RNA metabolism are spatially organized within the cell, we examined the subcellular distribution of a human DEAD box protein, DDX1, to identify possible biological functions. Immunofluorescence labeling of DDX1 demonstrated that in addition to widespread punctate nucleoplasmic labeling, DDX1 is found in discrete nuclear foci ∼0.5 μm in diameter. Costaining with anti-Sm and anti-promyelocytic leukemia (PML) antibodies indicates that DDX1 foci are frequently located next to Cajal (coiled) bodies and less frequently, to PML bodies. Most importantly, costaining with anti-CstF-64 antibody indicates that DDX1 foci colocalize with cleavage bodies. By microscopic fluorescence resonance energy transfer, we show that labeled DDX1 resides within a Förster distance of 10 nm of labeled CstF-64 protein in both the nucleoplasm and within cleavage bodies. Coimmunoprecipitation analysis indicates that a proportion of CstF-64 protein resides in the same complex as DDX1. These studies are the first to identify a DEAD box protein associating with factors involved in 3′-end cleavage and polyadenylation of pre-mRNAs.
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Gross, Stefan, and Claire L. Moore. "Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation." Molecular and Cellular Biology 21, no. 23 (December 1, 2001): 8045–55. http://dx.doi.org/10.1128/mcb.21.23.8045-8055.2001.
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ABSTRACT In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3′ end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3′ end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.
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Kaczmarek Michaels, Katarzyna, Salwa Mohd Mostafa, Julia Ruiz Capella, and Claire L. Moore. "Regulation of alternative polyadenylation in the yeast Saccharomyces cerevisiae by histone H3K4 and H3K36 methyltransferases." Nucleic Acids Research 48, no. 10 (May 1, 2020): 5407–25. http://dx.doi.org/10.1093/nar/gkaa292.
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Abstract Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3′ end processing machinery to the vicinity of pA sites.
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Calzado, Marco A., Rocío Sancho, and Eduardo Muñoz. "Human Immunodeficiency Virus Type 1 Tat Increases the Expression of Cleavage and Polyadenylation Specificity Factor 73-Kilodalton Subunit Modulating Cellular and Viral Expression." Journal of Virology 78, no. 13 (July 1, 2004): 6846–54. http://dx.doi.org/10.1128/jvi.78.13.6846-6854.2004.
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ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3′-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.
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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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So, Byung Ran, Chao Di, Zhiqiang Cai, Christopher C. Venters, Jiannan Guo, Jung-Min Oh, Chie Arai, and Gideon Dreyfuss. "A Complex of U1 snRNP with Cleavage and Polyadenylation Factors Controls Telescripting, Regulating mRNA Transcription in Human Cells." Molecular Cell 76, no. 4 (November 2019): 590–99. http://dx.doi.org/10.1016/j.molcel.2019.08.007.
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Holmes, Michael J., Leah R. Padgett, Matheus S. Bastos, and William J. Sullivan. "m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii." PLOS Pathogens 17, no. 7 (July 29, 2021): e1009335. http://dx.doi.org/10.1371/journal.ppat.1009335.
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Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3’-boundary of mRNAs. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3’-end processing of pre-mRNAs. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3’-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3’-ends.
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Amrani, N., M. Minet, F. Wyers, M. E. Dufour, L. P. Aggerbeck, and F. Lacroute. "PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I." Molecular and Cellular Biology 17, no. 3 (March 1997): 1102–9. http://dx.doi.org/10.1128/mcb.17.3.1102.
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Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing. Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified. We describe here another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I. This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15. PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa. Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15. The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15. Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation. Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody.
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Wong, Chi-Ming, Hongfang Qiu, Cuihua Hu, Jinsheng Dong, and Alan G. Hinnebusch. "Yeast Cap Binding Complex Impedes Recruitment of Cleavage Factor IA to Weak Termination Sites." Molecular and Cellular Biology 27, no. 18 (July 16, 2007): 6520–31. http://dx.doi.org/10.1128/mcb.00733-07.
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ABSTRACT Nuclear cap binding complex (CBC) is recruited cotranscriptionally and stimulates spliceosome assembly on nascent mRNAs; however, its possible functions in regulating transcription elongation or termination were not well understood. We show that, while CBC appears to be dispensable for normal rates and processivity of elongation by RNA polymerase II (Pol II), it plays a direct role in preventing polyadenylation at weak termination sites. Similarly to Npl3p, with which it interacts, CBC suppresses the weak terminator of the gal10-Δ56 mutant allele by impeding recruitment of termination factors Pcf11p and Rna15p (subunits of cleavage factor IA [CF IA]) and does so without influencing Npl3p occupancy at the termination site. Importantly, deletion of CBC subunits or NPL3 also increases termination at a naturally occurring weak poly(A) site in the RNA14 coding sequences. We also show that CBC is most likely recruited directly to the cap of nascent transcripts rather than interacting first with transcriptional activators or the phosphorylated C-terminal domain of Pol II. Thus, our findings illuminate the mechanism of CBC recruitment and extend its function in Saccharomyces cerevisiae beyond mRNA splicing and degradation of aberrant nuclear mRNAs to include regulation of CF IA recruitment at poly(A) selection sites.
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Dehm, Scott, Jeffery Miller, Sarah Munro, Jamie Van Etten, and Kiel Tietz. "OR26-1 Alternative Polyadenylation as a Therapeutic Vulnerability in Prostate Cancer." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A725. http://dx.doi.org/10.1210/jendso/bvac150.1494.
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Abstract Prostate cancer is the second leading cause of male cancer death in the United States. While localized disease can be cured by radiation or surgery, metastatic prostate cancer presents a clinical challenge. Metastatic prostate cancer can initially be controlled by endocrine therapies that target the androgen receptor (AR), however, these tumors will inevitably develop resistance. This stage of the disease, termed castration-resistant prostate cancer (CRPC), is responsible for the majority of prostate cancer-specific deaths. Truncated AR variant (AR-V) proteins are broadly enriched in CRPC cell lines and clinical samples, and can function as ligand-independent, constitutively active transcription factors. We found that blocking an alternative poly(A) site located in AR intron 3 reduced expression of multiple AR-V mRNA and protein species and increased expression of full-length (FL) AR mRNA and protein in 22Rv1 and LNCaP95 CRPC cells. We found the cleavage and polyadenylation specificity factor (CPSF) component, CPSF1, regulates selection of this alternative poly(A) site based on the finding that knockdown of CPSF1 also reduced expression of AR-Vs and increased expression of FL-AR in 22Rv1 and LNCaP95 cells. Further, knockdown of CPSF1 inhibited growth of these CRPC cell lines, as well as androgen-dependent LNCaP cells. To define how CPSF1 regulates alternative polyadenylation of AR and to determine novel pathways that are manipulated by CPSF1 in prostate cancer, we identified CPSF1-dependent gene expression using RNA-seq and CPSF1-dependent poly(A) site usage using poly(A)-ClickSeq (PAC-Seq). Gene set enrichment analysis of RNA-seq data revealed the Glycolysis Hallmark gene was positively regulated by CPSF1, and nominated multiple differentially-expressed genes encoding regulators of glycolysis as CPSF1 targets. These results highlight AR independent pathways that are also regulated by CPSF1 in prostate cancer. We are currently integrating RNA-Seq and PAC-seq datasets to define novel CPSF1 regulated pathways that can be targeted therapeutically in prostate cancer. This research will determine trans-acting factors and cis-regulatory elements that drive alternative polyadenylation of AR mRNA transcripts to promote CRPC progression. This research will also identify AR independent targets of CPSF1 that could be exploited for development of new therapies for CRPC. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.
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Waithaka, Albina, Olena Maiakovska, Dirk Grimm, Larissa Melo do Nascimento, and Christine Clayton. "Sequences and proteins that influence mRNA processing in Trypanosoma brucei: Evolutionary conservation of SR-domain and PTB protein functions." PLOS Neglected Tropical Diseases 16, no. 10 (October 26, 2022): e0010876. http://dx.doi.org/10.1371/journal.pntd.0010876.
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Background Spliced leader trans splicing is the addition of a short, capped sequence to the 5’ end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5’ trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA, which is a validated drug target. Methodology and principal findings We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by an intergenic region secondary structure. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. Conclusions Our selectable constructs are suitable for screens of both sequences, and proteins that affect mRNA processing in T. brucei. Our results suggest that the functions of PTB and SR-domain proteins in splice site definition may already have been present in the last eukaryotic common ancestor tract binding protein, SR-domain protein.
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Vagner, Stéphan, Christine Vagner, and Iain W. Mattaj. "The carboxyl terminus of vertebrate poly(A) polymerase interacts with U2AF 65 to couple 3′-end processing and splicing." Genes & Development 14, no. 4 (February 15, 2000): 403–13. http://dx.doi.org/10.1101/gad.14.4.403.
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Although it has been established that the processing factors involved in pre-mRNA splicing and 3′-end formation can influence each other positively, the molecular basis of this coupling interaction was not known. Stimulation of pre-mRNA splicing by an adjacentcis-linked cleavage and polyadenylation site in HeLa cell nuclear extract is shown to occur at an early step in splicing, the binding of U2AF 65 to the pyrimidine tract of the intron 3′ splice site. The carboxyl terminus of poly(A) polymerase (PAP) previously has been implicated indirectly in the coupling process. We demonstrate that a fusion protein containing the 20 carboxy-terminal amino acids of PAP, when tethered downstream of an intron, increases splicing efficiency and, like the entire 3′-end formation machinery, stimulates U2AF 65 binding to the intron. The carboxy-terminal domain of PAP makes a direct and specific interaction with residues 17–47 of U2AF 65, implicating this interaction in the coupling of splicing and 3′-end formation.
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Garrido-Lecca, Alfonso, and Thomas Blumenthal. "RNA Polymerase II C-Terminal Domain Phosphorylation Patterns in Caenorhabditis elegans Operons, Polycistronic Gene Clusters with Only One Promoter." Molecular and Cellular Biology 30, no. 15 (May 24, 2010): 3887–93. http://dx.doi.org/10.1128/mcb.00325-10.
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ABSTRACT The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5′ ends and serine 2 near 3′ ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5′ ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3′ ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3′ end by the usual mechanism. Although downstream operon genes have 5′ ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5′ ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3′ end, where the 3′-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.
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Ruta, Veronica, Vittoria Pagliarini, and Claudio Sette. "Coordination of RNA Processing Regulation by Signal Transduction Pathways." Biomolecules 11, no. 10 (October 7, 2021): 1475. http://dx.doi.org/10.3390/biom11101475.
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Signal transduction pathways transmit the information received from external and internal cues and generate a response that allows the cell to adapt to changes in the surrounding environment. Signaling pathways trigger rapid responses by changing the activity or localization of existing molecules, as well as long-term responses that require the activation of gene expression programs. All steps involved in the regulation of gene expression, from transcription to processing and utilization of new transcripts, are modulated by multiple signal transduction pathways. This review provides a broad overview of the post-translational regulation of factors involved in RNA processing events by signal transduction pathways, with particular focus on the regulation of pre-mRNA splicing, cleavage and polyadenylation. The effects of several post-translational modifications (i.e., sumoylation, ubiquitination, methylation, acetylation and phosphorylation) on the expression, subcellular localization, stability and affinity for RNA and protein partners of many RNA-binding proteins are highlighted. Moreover, examples of how some of the most common signal transduction pathways can modulate biological processes through changes in RNA processing regulation are illustrated. Lastly, we discuss challenges and opportunities of therapeutic approaches that correct RNA processing defects and target signaling molecules.
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Su, Yan, Richard Adair, Candice N. Davis, Nancy L. DiFronzo, and Anamaris M. Colberg-Poley. "Convergence of RNA cis Elements and Cellular Polyadenylation Factors in the Regulation of Human Cytomegalovirus UL37 Exon 1 Unspliced RNA Production." Journal of Virology 77, no. 23 (December 1, 2003): 12729–41. http://dx.doi.org/10.1128/jvi.77.23.12729-12741.2003.
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ABSTRACT The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for its growth. The UL37 promoter drives production of an unspliced and several alternatively spliced RNAs. The UL37 exon 1 (UL37x1) unspliced RNA is abundant from IE to late times of HCMV infection, whereas the UL37 spliced RNAs are markedly less abundant. Production of the UL37x1 unspliced RNA requires polyadenylation (PA) at nucleotide 50998, which lies within intron 1, upstream of the UL37 exon 2 (UL37x2) acceptor. The physical proximity of its cis elements suggests steric hindrance between PA and splicing machineries for UL37 pre-mRNA. To test this possibility, we generated site-specific mutants in Target 1 PA and RNA splicing cis elements and compared the PA and splicing efficiencies of mutant RNAs with those of wild-type RNA. The mutually exclusive processing events of UL37x1 PA and UL37x1-UL37x2 splicing have been accurately recapitulated in transfected permissive human fibroblasts (HFFs) expressing a Target 1 minigene RNA, which contains the required splicing and PA cis elements. Two mutants in the invariant PA signal dramatically decreased UL37x1 PA as expected and, concomitantly, increased the efficiency of UL37x1-UL37x2 RNA splicing. Consistent with these results, changes to consensus UL37x1 donor and UL37x2 acceptor sites increased the efficiency of UL37x1-UL37x2 RNA splicing but decreased the efficiency of UL37x1 PA. Moreover, HCMV infection of HFFs increased the abundance of the PA cleavage stimulatory factor CstF-64, the potent splicing suppressor PTB, and the hypophosphorylated form of the splicing factor SF2 at 4 h postinfection. Induction of these factors further favors production of the UL37x1 unspliced RNA over that of the spliced RNAs. Taken together, these results suggest that there is a convergence in UL37 RNA regulation by cis elements and cellular proteins which favors production of the UL37x1 unspliced RNA during HCMV infection at the posttranscriptional level.
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Gall, Joseph G., Michel Bellini, Zheng’an Wu, and Christine Murphy. "Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes." Molecular Biology of the Cell 10, no. 12 (December 1999): 4385–402. http://dx.doi.org/10.1091/mbc.10.12.4385.
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We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.
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Magrath, Christi, and Linda E. Hyman. "A Mutation in GRS1, a Glycyl-tRNA Synthetase, Affects 3′-End Formation in Saccharomyces cerevisiae." Genetics 152, no. 1 (May 1, 1999): 129–41. http://dx.doi.org/10.1093/genetics/152.1.129.
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Abstract 3′-end formation is a complex and incompletely understood process involving both cis-acting and trans-acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a mutant hunt for strains defective in 3′-end formation was conducted. Following random mutagenesis, a temperature-sensitive strain exhibiting several phenotypes consistent with a role in transcription termination was isolated. First, readthrough of a terminator increases significantly in the mutant strain. Accordingly, RNA analysis indicates a decrease in the level of terminated transcripts, both in vivo and in vitro. Moreover, a plasmid stability assay in which high levels of readthrough lead to high levels of plasmid loss and transcription run-on analysis also demonstrate defective termination of transcription. Examination of polyadenylation and cleavage by the mutant strain indicates these processes are not affected. These results represent the first example of a transcription termination factor in Saccharomyces cerevisiae that affects transcription termination independent of 3′-end processing of mRNA. Complementation studies identified GRS1, an aminoacyl-tRNA synthetase, as the complementing gene. Sequence analysis of grs1-1 in the mutant strain revealed that nucleotides 1656 and 1657 were both C to T transitions, resulting in a single amino acid change of proline to phenylalanine. Further studies revealed GRS1 is essential, and the grs1-1 allele confers the temperature-sensitive growth defect associated with the mutant strain. Finally, we observed structures with some similarity to tRNA molecules within the 3′-end of various yeast genes. On the basis of our results, we suggest Grs1p is a transcription termination factor that may interact with the 3′-end of pre-mRNA to promote 3′-end formation.
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Bolli, Niccolo, Elspeth Payne, Jennifer Rhodes, Adam Johnston, Feng Guo, Jeong-Soo Lee, John Kanki, et al. "Cleavage and Polyadenylation Specificity Factor 1 Is Required for Definitive Hematopoietic Stem Cell Survival In Zebrafish." Blood 116, no. 21 (November 19, 2010): 1606. http://dx.doi.org/10.1182/blood.v116.21.1606.1606.
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Abstract Abstract 1606 Hematopoiesis is a tightly regulated process requiring the differential activation of specific genetic programs. Inactivating mutations of genes involved in normal hematopoiesis have been implicated in bone marrow failure syndromes, myelodysplastic syndromes and hematopoietic neoplasms. Despite recent advances, many genes regulating normal hematopoiesis are still unknown and innovative gene-discovery approaches can benefit our understanding of the molecular mechanisms underlying this complex developmental process. The combined genetic and embryologic strengths of the zebrafish model system are ideal for studying factors regulating hematopoiesis, which is highly conserved with mammals. We have used ENU-mutated zebrafish lines to screen for genes whose inactivation results in the loss of definitive hematopoiesis. Here, we report the identification and analysis of the zebrafish mutant grechetto, caused by a lethal, recessive, inactivating mutation (grcl8a12) of the cleavage and specificity factor 1 (cpsf1) gene. Cpsf1 encodes a protein required for processing the 3’UTR of a subset of pre-mRNAs. Cpsf1 is maternally expressed ubiquitously through the first five days of development including primitive myeloid and erythroid cells and definitive hematopoietic stem cells (HSC). Although grechetto mutants fail to express cspf1 already at 24 hpf, they appear to develop normally until 72 hpf, when they first become morphologically distinct from wild-type siblings with a smaller head and the absence of a protruding jaw. Importantly, primitive hematopoiesis appears normal, as shown by expression of the myeloid and erythroid markers mpx and band3, respectively, at 24 and 48 hpf by whole mount RNA in situ hybridization (WISH). By 120 hpf the phenotype is more marked and embryos have a curved body, cardiac edema, development defects in the jaw and gut and a reduction in the number of iridophores. While many of these affected tissues represent derivatives of the neural crest (NC), the expression of markers of NC specification (sox10 and foxd3 at 15,5 hpf, crestin at 24 hpf), migration (dlx2 at 36 and 48 hpf) and differentiation (pax9a at 48 hpf) are normal. When definitive hematopoiesis was assayed for markers of mature blood cells by WISH at 120 hpf, grechetto mutants exhibit a loss of myeloid (mpx, lysC, l-plastin), erythroid (band3, gata1) and lymphoid cells (rag1, lck). Since myeloid and erythroid cells are normal at 24 hpf, but severely reduced at 120 hpf, we investigated whether the absence of HSC could explain the loss of definitive hematopoiesis in grechetto mutants. Interestingly, WISH staining for the HSC marker c-myb showed that in mutant embryos HSCs are normally specified and represented in the ventral wall of the dorsal aorta at 36 and 48 hpf. Nevertheless, at 72 hpf the numbers of HSCs are reduced in grechetto mutants upon their migration to the caudal hematopoietic tissue (CHT) and by 120 hpf very few remain. To investigate the fate of these HSCs we crossed the grechetto line with the Tg(c-myb:EGFP) reporter line, that expresses EGFP under the control of the c-myb promoter. By whole mount immunostaining, we were able to find increased co-localization of activated caspase3 and TUNEL staining in the c-myb:EGFP positive cells in the CHT in grechetto mutants compared to WT siblings at 72 and 96 hpf, suggesting that HSCs undergo apoptosis during this stage. Furthermore, PI cell cycle profile of sorted c-myb:EGFP+ cells from 96 hpf mutants shows a sub-G1 peak, representing pyknotic cell nuclei undergoing advanced stages of apoptosis. Importantly, both activated caspase3 staining and TUNEL assay did not show increased apoptosis in the tissue immediately surrounding the CHT. These studies show that cpsf1 is not required for primitive hematopoiesis or definitive HSC specification, however, it is essential for HSC survival in the CHT. The fact that grechetto embryos do not undergo generalized apoptotic cell death suggests that, despite ubiquitous cpsf1 expression and the general development defects of grechetto mutants, the prosurvival pathways in the HSC compartment are particularly dependent on Cpsf1 activity. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.