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Journal articles on the topic 'MRNA report'
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1
Shen, Qingtang, Yifan E. Wang, Mathew Truong, Kohila Mahadevan, Jingze J. Wu, Hui Zhang, Jiawei Li, Harrison W. Smith, Craig A. Smibert, and Alexander F. Palazzo. "RanBP2/Nup358 enhances miRNA activity by sumoylating Argonautes." PLOS Genetics 17, no. 2 (February 18, 2021): e1009378. http://dx.doi.org/10.1371/journal.pgen.1009378.
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Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.
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Mili, Stavroula, Hong Jun Shu, Yingming Zhao, and Serafı́n Piñol-Roma. "Distinct RNP Complexes of Shuttling hnRNP Proteins with Pre-mRNA and mRNA: Candidate Intermediates in Formation and Export of mRNA." Molecular and Cellular Biology 21, no. 21 (November 1, 2001): 7307–19. http://dx.doi.org/10.1128/mcb.21.21.7307-7319.2001.
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ABSTRACT Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.
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Meaux, Stacie A., Christopher E. Holmquist, and William F. Marzluff. "Role of oligouridylation in normal metabolism and regulated degradation of mammalian histone mRNAs." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1762 (November 5, 2018): 20180170. http://dx.doi.org/10.1098/rstb.2018.0170.
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Metazoan replication-dependent histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Histone mRNAs are present in large amounts only in S-phase cells, and their levels are coordinately regulated with the rate of DNA replication. In mammals, the stemloop at the 3′ end of histone mRNA is bound to stemloop binding protein, a protein required for both synthesis and degradation of histone mRNA, and an exonuclease, 3′hExo (ERI1). Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S-phase cells and at the end of S-phase. Upf1 is also required for rapid degradation of histone mRNA as is the S-phase checkpoint. We report that Smg1 is required for histone mRNA degradation when DNA replication is inhibited, suggesting it is the PI-like kinase that activates Upf1 for histone mRNA degradation. We also show that some mutant Upf1 proteins are recruited to histone mRNAs when DNA replication is inhibited and act as dominant negative factors in histone mRNA degradation. We report that the pathway of rapid histone mRNA degradation when DNA replication is inhibited in S-phase cells that are activating the S-phase checkpoint is similar to the pathway of rapid degradation of histone mRNA at the end of S-phase. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.
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Kong, Jian, Marina Sumaroka, Dawn L. Eastmond, and Stephen A. Liebhaber. "Shared Stabilization Functions of Pyrimidine-Rich Determinants in the Erythroid 15-lipoxygenase and α-globin mRNAs." Molecular and Cellular Biology 26, no. 15 (August 1, 2006): 5603–14. http://dx.doi.org/10.1128/mcb.01845-05.
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ABSTRACT The poly(C)-binding proteins, αCPs, comprise a set of highly conserved KH-domain factors that participate in mRNA stabilization and translational controls in developmental and viral systems. Two prominent models of αCP function link these controls to late stages of erythroid differentiation: translational silencing of 15-lipoxygenase (Lox) mRNA and stabilization of α-globin mRNA. These two controls are mediated via association of αCPs with structurally related C-rich 3′-untranslated region elements: the differentiation control elements (DICE) in Lox mRNA and the pyrimidine-rich motifs in α-globin mRNA. In the present report a set of mRNA translation and stability assays are used to determine how these two αCP-containing complexes, related in structure and position, mediate distinct posttranscriptional controls. While the previously reported translational silencing by the DICE is not evident in our studies, we find that the two determinants mediate similar levels of mRNA stabilization in erythroid cells. In both cases this stabilization is sensitive to interference by a nuclear-restricted αCP decoy but not by the same decoy restricted to the cytoplasm. These data support a general role for αCPs in stabilizing a subset of erythroid mRNAs. The findings also suggest that initial binding of αCP to target mRNAs occurs in the nucleus. Assembly of stabilizing mRNP complexes in the nucleus prior to export may maximize their impact on cytoplasmic events.
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Corcoran, Jennifer A., Wei-Li Hsu, and James R. Smiley. "Herpes Simplex Virus ICP27 Is Required for Virus-Induced Stabilization of the ARE-Containing IEX-1 mRNA Encoded by the Human IER3 Gene." Journal of Virology 80, no. 19 (October 1, 2006): 9720–29. http://dx.doi.org/10.1128/jvi.01216-06.
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ABSTRACT Herpes simplex virus (HSV) stifles cellular gene expression during productive infection of permissive cells, thereby diminishing host responses to infection. Host shutoff is achieved largely through the complementary actions of two viral proteins, ICP27 and virion host shutoff (vhs), that inhibit cellular mRNA biogenesis and trigger global mRNA decay, respectively. Although most cellular mRNAs are thus depleted, some instead increase in abundance after infection; perhaps surprisingly, some of these contain AU-rich instability elements (AREs) in their 3′-untranslated regions. ARE-containing mRNAs normally undergo rapid decay; however, their stability can increase in response to signals such as cytokines and virus infection that activate the p38/MK2 mitogen-activated protein kinase (MAPK) pathway. We and others have shown that HSV infection stabilizes the ARE mRNA encoding the stress-inducible IEX-1 mRNA, and a previous report from another laboratory has suggested vhs is responsible for this effect. However, we now report that ICP27 is essential for IEX-1 mRNA stabilization whereas vhs plays little if any role. A recent report has documented that ICP27 activates the p38 MAPK pathway, and we detected a strong correlation between this activity and stabilization of IEX-1 mRNA by using a panel of HSV type 1 (HSV-1) isolates bearing an array of previously characterized ICP27 mutations. Furthermore, IEX-1 mRNA stabilization was abrogated by the p38 inhibitor SB203580. Taken together, these data indicate that the HSV-1 immediate-early protein ICP27 alters turnover of the ARE-containing message IEX-1 by activating p38. As many ARE mRNAs encode proinflammatory cytokines or other immediate-early response proteins, some of which may limit viral replication, it will be of great interest to determine if ICP27 mediates stabilization of many or all ARE-containing mRNAs.
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Hsu, Wei-Li, Holly A. Saffran, and James R. Smiley. "Herpes Simplex Virus Infection Stabilizes Cellular IEX-1 mRNA." Journal of Virology 79, no. 7 (April 1, 2005): 4090–98. http://dx.doi.org/10.1128/jvi.79.7.4090-4098.2005.
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ABSTRACT Herpes simplex virus (HSV) virion host shutoff protein (vhs) destabilizes cellular and viral mRNAs. Previous work from several laboratories has indicated that vhs accelerates the turnover of most host mRNAs and provided evidence that at least some of these are degraded via endonucleolytic cleavage near regions of translational initiation followed by 5′→3′ decay. In contrast, several recent reports have argued that vhs is selective, preferentially targeting a subset of mRNAs including some that bear AU-rich instability elements (such as the stress-inducible IEX-1 mRNA). These reports concluded that vhs triggers deadenylation, 3′ cleavage, and 3′→5′ decay of IEX-1 mRNA. However, we report here that HSV infection does not increase the rate of degradation of IEX-1 mRNA; rather, actinomycin D chase assays indicate that the transcript is stabilized relative to that in uninfected cells in both the presence and absence of functional vhs. Moreover, deadenylated but otherwise intact IEX-1 mRNA was readily detected in uninfected cells cultured under our experimental conditions, and its relative abundance did not increase following HSV type 1 (HSV-1) infection. We confirm that HSV infection increases the relative abundance of a discrete 0.75-kb 3′-truncated IEX-1 RNA species in a vhs-dependent manner. This truncated transcript was also detected (albeit at lower levels) in cells infected with vhs mutants and in uninfected cells, where it increased in abundance in response to tumor necrosis factor alpha, cycloheximide, and puromycin. We conclude that IEX-1 mRNA is not preferentially degraded during HSV-1 infection and that HSV-1 instead inhibits the normal turnover of this mRNA.
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Kay, M. A., and M. Jacobs-Lorena. "Selective translational regulation of ribosomal protein gene expression during early development of Drosophila melanogaster." Molecular and Cellular Biology 5, no. 12 (December 1985): 3583–92. http://dx.doi.org/10.1128/mcb.5.12.3583.
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We have previously characterized a cloned cDNA coding for a developmentally regulated mRNA in Drosophila melanogaster whose expression is selectively regulated at the translational level during oogenesis and embryogenesis. In this report we show that this translationally regulated mRNA (rpA1) codes for an acidic ribosomal protein. Furthermore, our results indicate that most ribosomal protein mRNAs are regulated similarly to rpA1 mRNA. This conclusion is based on cell-free translation of mRNAs derived from polysomes and postpolysomal supernatants as well as in vivo labeling experiments. Thus, the translation of many ribosomal protein mRNAs appears to be temporally related to the synthesis of rRNA during D. melanogaster development. The relationship between rRNA transcription and ribosomal protein mRNA translation was further investigated by genetically reducing rRNA synthesis with the use of bobbed mutants. Unexpectedly, neither ribosomal protein mRNA abundance nor translation was altered in these mutants.
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Kay, M. A., and M. Jacobs-Lorena. "Selective translational regulation of ribosomal protein gene expression during early development of Drosophila melanogaster." Molecular and Cellular Biology 5, no. 12 (December 1985): 3583–92. http://dx.doi.org/10.1128/mcb.5.12.3583-3592.1985.
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We have previously characterized a cloned cDNA coding for a developmentally regulated mRNA in Drosophila melanogaster whose expression is selectively regulated at the translational level during oogenesis and embryogenesis. In this report we show that this translationally regulated mRNA (rpA1) codes for an acidic ribosomal protein. Furthermore, our results indicate that most ribosomal protein mRNAs are regulated similarly to rpA1 mRNA. This conclusion is based on cell-free translation of mRNAs derived from polysomes and postpolysomal supernatants as well as in vivo labeling experiments. Thus, the translation of many ribosomal protein mRNAs appears to be temporally related to the synthesis of rRNA during D. melanogaster development. The relationship between rRNA transcription and ribosomal protein mRNA translation was further investigated by genetically reducing rRNA synthesis with the use of bobbed mutants. Unexpectedly, neither ribosomal protein mRNA abundance nor translation was altered in these mutants.
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Chen, Qiang, Sujatha Jagannathan, David W. Reid, Tianli Zheng, and Christopher V. Nicchitta. "Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells." Molecular Biology of the Cell 22, no. 14 (July 15, 2011): 2646–58. http://dx.doi.org/10.1091/mbc.e11-03-0239.
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The mRNA transcriptome is currently thought to be partitioned between the cytosol and endoplasmic reticulum (ER) compartments by binary selection; mRNAs encoding cytosolic/nucleoplasmic proteins are translated on free ribosomes, and mRNAs encoding topogenic signal-bearing proteins are translated on ER-bound ribosomes, with ER localization being conferred by the signal-recognition particle pathway. In subgenomic and genomic analyses of subcellular mRNA partitioning, we report an overlapping subcellular distribution of cytosolic/nucleoplasmic and topogenic signal-encoding mRNAs, with mRNAs of both cohorts displaying noncanonical subcellular partitioning patterns. Unexpectedly, the topogenic signal-encoding mRNA transcriptome was observed to partition in a hierarchical, cohort-specific manner. mRNAs encoding resident proteins of the endomembrane system were clustered at high ER-enrichment values, whereas mRNAs encoding secretory pathway cargo were broadly represented on free and ER-bound ribosomes. Two distinct modes of mRNA association with the ER were identified. mRNAs encoding endomembrane-resident proteins were bound via direct, ribosome-independent interactions, whereas mRNAs encoding secretory cargo displayed predominantly ribosome-dependent modes of ER association. These data indicate that mRNAs are partitioned between the cytosol and ER compartments via a hierarchical system of intrinsic and encoded topogenic signals and identify mRNA cohort-restricted modes of mRNA association with the ER.
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Proulex, Grayson C. R., Marcus J. Meade, Kalina M. Manoylov, and A. Bruce Cahoon. "Mitochondrial mRNA Processing in the Chlorophyte Alga Pediastrum duplex and Streptophyte Alga Chara vulgaris Reveals an Evolutionary Branch in Mitochondrial mRNA Processing." Plants 10, no. 3 (March 18, 2021): 576. http://dx.doi.org/10.3390/plants10030576.
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Mitochondria carry the remnant of an ancestral bacterial chromosome and express those genes with a system separate and distinct from the nucleus. Mitochondrial genes are transcribed as poly-cistronic primary transcripts which are post-transcriptionally processed to create individual translationally competent mRNAs. Algae post-transcriptional processing has only been explored in Chlamydomonas reinhardtii (Class: Chlorophyceae) and the mature mRNAs are different than higher plants, having no 5′ UnTranslated Regions (UTRs), much shorter and more variable 3′ UTRs and polycytidylated mature mRNAs. In this study, we analyzed transcript termini using circular RT-PCR and PacBio Iso-Seq to survey the 3′ and 5′ UTRs and termini for two green algae, Pediastrum duplex (Class: Chlorophyceae) and Chara vulgaris (Class: Charophyceae). This enabled the comparison of processing in the chlorophyte and charophyte clades of green algae to determine if the differences in mitochondrial mRNA processing pre-date the invasion of land by embryophytes. We report that the 5′ mRNA termini and non-template 3′ termini additions in P. duplex resemble those of C. reinhardtii, suggesting a conservation of mRNA processing among the chlorophyceae. We also report that C. vulgaris mRNA UTRs are much longer than chlorophytic examples, lack polycytidylation, and are polyadenylated similar to embryophytes. This demonstrates that some mitochondrial mRNA processing events diverged with the split between chlorophytic and streptophytic algae.
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Brady, H. A., and W. S. Wold. "Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized." Molecular and Cellular Biology 8, no. 8 (August 1988): 3291–97. http://dx.doi.org/10.1128/mcb.8.8.3291.
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Complex transcription units encode multiple mRNAs which arise by alternative processing of a common pre-mRNA precursor. It is not known how the pre-mRNA processing pathways are determined or controlled. We are investigating this problem by using the E3 complex transcription unit of adenovirus as a model. Our approach is to construct virus mutants with lesions in E3 and then determine how the mutation affects the accumulation of E3 mRNAs in vivo. We report results which indicate that competition between splicing reactions and polyadenylation reactions occurs in vivo and that this plays an important role in alternative pre-mRNA processing.
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Brady, H. A., and W. S. Wold. "Competition between splicing and polyadenylation reactions determines which adenovirus region E3 mRNAs are synthesized." Molecular and Cellular Biology 8, no. 8 (August 1988): 3291–97. http://dx.doi.org/10.1128/mcb.8.8.3291-3297.1988.
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Complex transcription units encode multiple mRNAs which arise by alternative processing of a common pre-mRNA precursor. It is not known how the pre-mRNA processing pathways are determined or controlled. We are investigating this problem by using the E3 complex transcription unit of adenovirus as a model. Our approach is to construct virus mutants with lesions in E3 and then determine how the mutation affects the accumulation of E3 mRNAs in vivo. We report results which indicate that competition between splicing reactions and polyadenylation reactions occurs in vivo and that this plays an important role in alternative pre-mRNA processing.
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Baumann, Sebastian, Artem Komissarov, Maria Gili, Verena Ruprecht, Stefan Wieser, and Sebastian P. Maurer. "A reconstituted mammalian APC-kinesin complex selectively transports defined packages of axonal mRNAs." Science Advances 6, no. 11 (March 2020): eaaz1588. http://dx.doi.org/10.1126/sciadv.aaz1588.
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Through the asymmetric distribution of messenger RNAs (mRNAs), cells spatially regulate gene expression to create cytoplasmic domains with specialized functions. In neurons, mRNA localization is required for essential processes such as cell polarization, migration, and synaptic plasticity underlying long-term memory formation. The essential components driving cytoplasmic mRNA transport in neurons and mammalian cells are not known. We report the first reconstitution of a mammalian mRNA transport system revealing that the tumor suppressor adenomatous polyposis coli (APC) forms stable complexes with the axonally localized β-actin and β2B-tubulin mRNAs, which are linked to a kinesin-2 via the cargo adaptor KAP3. APC activates kinesin-2, and both proteins are sufficient to drive specific transport of defined mRNA packages. Guanine-rich sequences located in 3′UTRs of axonal mRNAs increase transport efficiency and balance the access of different mRNAs to the transport system. Our findings reveal a minimal set of proteins sufficient to transport mammalian mRNAs.
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Svoboda, P., P. Stein, H. Hayashi, and R. M. Schultz. "Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference." Development 127, no. 19 (October 1, 2000): 4147–56. http://dx.doi.org/10.1242/dev.127.19.4147.
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Specific mRNA degradation mediated by double-stranded RNA (dsRNA), which is termed RNA interference (RNAi), is a useful tool with which to study gene function in several systems. We report here that in mouse oocytes, RNAi provides a suitable and robust approach to study the function of dormant maternal mRNAs. Mos (originally known as c-mos) and tissue plasminogen activator (tPA, Plat) mRNAs are dormant maternal mRNAs that are recruited during oocyte maturation; translation of Mos mRNA results in the activation of MAP kinase. dsRNA directed towards Mos or Plat mRNAs in mouse oocytes effectively results in the specific reduction of the targeted mRNA in both a time- and concentration-dependent manner. Moreover, dsRNA is more potent than either sense or antisense RNAs. Targeting the Mos mRNA results in inhibiting the appearance of MAP kinase activity and can result in parthenogenetic activation. Mos dsRNA, therefore, faithfully phenocopies the Mos null mutant. Targeting the Plat mRNA with Plat dsRNA results in inhibiting production of tPA activity. Finally, effective reduction of the Mos and Plat mRNA is observed with stoichiometric amounts of Mos and Plat dsRNA, respectively.
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Boeck, Ronald, Bruno Lapeyre, Christine E. Brown, and Alan B. Sachs. "Capped mRNA Degradation Intermediates Accumulate in the Yeast spb8-2 Mutant." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 5062–72. http://dx.doi.org/10.1128/mcb.18.9.5062.
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ABSTRACT mRNA in the yeast Saccharomyces cerevisiae is primarily degraded through a pathway that is stimulated by removal of the mRNA cap structure. Here we report that a mutation in the SPB8(YJL124c) gene, initially identified as a suppressor mutation of a poly(A)-binding protein (PAB1) gene deletion, stabilizes the mRNA cap structure. Specifically, we find that thespb8-2 mutation results in the accumulation of capped, poly(A)-deficient mRNAs. The presence of this mutation also allows for the detection of mRNA species trimmed from the 3′ end. These data show that this Sm-like protein family member is involved in the process of mRNA decapping, and they provide an example of 3′-5′ mRNA degradation intermediates in yeast.
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Kufel, Joanna, Cecile Bousquet-Antonelli, Jean D. Beggs, and David Tollervey. "Nuclear Pre-mRNA Decapping and 5′ Degradation in Yeast Require the Lsm2-8p Complex." Molecular and Cellular Biology 24, no. 21 (November 1, 2004): 9646–57. http://dx.doi.org/10.1128/mcb.24.21.9646-9657.2004.
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ABSTRACT Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes. Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p. These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover. Lsm8p can be UV cross-linked to nuclear poly(A)+ RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA. Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5′ degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p. Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in lsm mutants remain 5′ capped. We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.
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Brewer, G. "An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro." Molecular and Cellular Biology 11, no. 5 (May 1991): 2460–66. http://dx.doi.org/10.1128/mcb.11.5.2460.
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Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.
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Brewer, G. "An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro." Molecular and Cellular Biology 11, no. 5 (May 1991): 2460–66. http://dx.doi.org/10.1128/mcb.11.5.2460-2466.1991.
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Transient expression of some proto-oncogenes, cytokines, and transcription factors occurs as a cellular response to growth factors, 12-O-tetradecanoylphorbol-13-acetate, antigen stimulation, or inflammation. Expression of these genes is mediated in part by the rapid turnover of their mRNAs. A + U-rich elements in the 3' untranslated regions of these mRNAs serve as one recognition signal targeting the mRNAs for rapid degradation. I report the identification of a cytosolic factor that both binds to the proto-oncogene c-myc A + U-rich element and specifically destabilizes c-myc mRNA in a cell-free mRNA decay system which reconstitutes mRNA decay processes found in cells. Proteinase K treatment of the factor abolishes its c-myc mRNA degradation activity without affecting its RNA-binding capacity. Thus, RNA substrate binding and degradation appear to be separable functions. These findings should aid in understanding how the cell selectively targets mRNAs for rapid turnover.
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Paul, Biplab, and Ben Montpetit. "Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus." Molecular Biology of the Cell 27, no. 17 (September 2016): 2742–56. http://dx.doi.org/10.1091/mbc.e16-04-0244.
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Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3′-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus.
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Jackson, Lisa A., Evan J. Anderson, Nadine G. Rouphael, Paul C. Roberts, Mamodikoe Makhene, Rhea N. Coler, Michele P. McCullough, et al. "An mRNA Vaccine against SARS-CoV-2 — Preliminary Report." New England Journal of Medicine 383, no. 20 (November 12, 2020): 1920–31. http://dx.doi.org/10.1056/nejmoa2022483.
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Zimak, Jan, Yonglei Shang, and Weian Zhao. "mRNA rescues neonatal acidemia while mice report no aftereffects." EBioMedicine 46 (August 2019): 23–24. http://dx.doi.org/10.1016/j.ebiom.2019.07.064.
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Keeling, Kim M., Joe Salas-Marco, Lev Z. Osherovich, and David M. Bedwell. "Tpa1p Is Part of an mRNP Complex That Influences Translation Termination, mRNA Deadenylation, and mRNA Turnover in Saccharomyces cerevisiae." Molecular and Cellular Biology 26, no. 14 (July 15, 2006): 5237–48. http://dx.doi.org/10.1128/mcb.02448-05.
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ABSTRACT In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for termination and polyadenylation) influences translation termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1Δ) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the translation termination factors eRF1 and eRF3. In addition, the tpa1Δ mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 5′→3′ pathway or the 3′→5′ nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1Δ strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3′ untranslated region of mRNAs that affects translation termination, deadenylation, and mRNA decay.
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Chuang, Tzu-Wei, Wei-Lun Chang, Kuo-Ming Lee, and Woan-Yuh Tarn. "The RNA-binding protein Y14 inhibits mRNA decapping and modulates processing body formation." Molecular Biology of the Cell 24, no. 1 (January 2013): 1–13. http://dx.doi.org/10.1091/mbc.e12-03-0217.
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The exon-junction complex (EJC) deposited on a newly spliced mRNA plays an important role in subsequent mRNA metabolic events. Here we show that an EJC core heterodimer, Y14/Magoh, specifically associates with mRNA-degradation factors, including the mRNA-decapping complex and exoribonucleases, whereas another core factor, eIF4AIII/MLN51, does not. We also demonstrate that Y14 interacts directly with the decapping factor Dcp2 and the 5′ cap structure of mRNAs via different but overlapping domains and that Y14 inhibits the mRNA-decapping activity of Dcp2 in vitro. Accordingly, overexpression of Y14 prolongs the half-life of a reporter mRNA. Therefore Y14 may function independently of the EJC in preventing mRNA decapping and decay. Furthermore, we observe that depletion of Y14 disrupts the formation of processing bodies, whereas overexpression of a phosphomimetic Y14 considerably increases the number of processing bodies, perhaps by sequestering the mRNA-degradation factors. In conclusion, this report provides unprecedented evidence for a role of Y14 in regulating mRNA degradation and processing body formation and reinforces the influence of phosphorylation of Y14 on its activity in postsplicing mRNA metabolism.
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Roth, Kelly M., Maria K. Wolf, Marie Rossi, and J. Scott Butler. "The Nuclear Exosome Contributes to Autogenous Control of NAB2 mRNA Levels." Molecular and Cellular Biology 25, no. 5 (March 1, 2005): 1577–85. http://dx.doi.org/10.1128/mcb.25.5.1577-1585.2005.
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ABSTRACT The RNA-processing exosome is a complex of riboexonucleases required for 3′-end formation of some noncoding RNAs and for the degradation of mRNAs in eukaryotes. The nuclear form of the exosome functions in an mRNA surveillance pathway that retains and degrades improperly processed precursor mRNAs within the nucleus. We report here that the nuclear exosome controls the level of NAB2 mRNA, encoding the nuclear poly(A)+-RNA-binding protein Nab2p. Mutations affecting the activity of the nuclear, but not the cytoplasmic, exosome cause an increase in the amount of NAB2 mRNA. Cis- and trans-acting mutations that inhibit degradation by the nuclear-exosome subunit Rrp6p result in elevated levels of NAB2 mRNA. Control of NAB2 mRNA levels occurs posttranscriptionally and requires a sequence of 26 consecutive adenosines (A26) in the NAB2 3′ untranslated region, which represses NAB2 3′-end formation and sensitizes the transcript to degradation by Rrp6p. Analysis of NAB2 mRNA levels in a nab2-1 mutant and in the presence of excess Nab2p indicates that Nab2p activity negatively controls NAB2 mRNA levels in an A26- and Rrp6p-dependent manner. These findings suggest a novel regulatory circuit in which the nuclear exosome controls the level of NAB2 mRNA in response to changes in the activity of Nab2 protein.
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Charenton, Clément, Claudine Gaudon-Plesse, Régis Back, Nathalie Ulryck, Loreline Cosson, Bertrand Séraphin, and Marc Graille. "Pby1 is a direct partner of the Dcp2 decapping enzyme." Nucleic Acids Research 48, no. 11 (May 12, 2020): 6353–66. http://dx.doi.org/10.1093/nar/gkaa337.
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Abstract Most eukaryotic mRNAs harbor a characteristic 5′ m7GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5′-3′ exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1–Dcp2–Edc3 decapping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation, both stemming from its interaction with the Dcp1–Dcp2 holoenzyme.
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Burgui, Idoia, Emilio Yángüez, Nahum Sonenberg, and Amelia Nieto. "Influenza Virus mRNA Translation Revisited: Is the eIF4E Cap-Binding Factor Required for Viral mRNA Translation?" Journal of Virology 81, no. 22 (September 12, 2007): 12427–38. http://dx.doi.org/10.1128/jvi.01105-07.
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ABSTRACT Influenza virus mRNAs bear a short capped oligonucleotide sequence at their 5′ ends derived from the host cell pre-mRNAs by a “cap-snatching” mechanism, followed immediately by a common viral sequence. At their 3′ ends, they contain a poly(A) tail. Although cellular and viral mRNAs are structurally similar, influenza virus promotes the selective translation of its mRNAs despite the inhibition of host cell protein synthesis. The viral polymerase performs the cap snatching and binds selectively to the 5′ common viral sequence. As viral mRNAs are recognized by their own cap-binding complex, we tested whether viral mRNA translation occurs without the contribution of the eIF4E protein, the cellular factor required for cap-dependent translation. Here, we show that influenza virus infection proceeds normally in different situations of functional impairment of the eIF4E factor. In addition, influenza virus polymerase binds to translation preinitiation complexes, and furthermore, under conditions of decreased eIF4GI association to cap structures, an increase in eIF4GI binding to these structures was found upon influenza virus infection. This is the first report providing evidence that influenza virus mRNA translation proceeds independently of a fully active translation initiation factor (eIF4E). The data reported are in agreement with a role of viral polymerase as a substitute for the eIF4E factor for viral mRNA translation.
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Stephens, Samuel B., Rebecca D. Dodd, Joseph W. Brewer, Patrick J. Lager, Jack D. Keene, and Christopher V. Nicchitta. "Stable Ribosome Binding to the Endoplasmic Reticulum Enables Compartment-specific Regulation of mRNA Translation." Molecular Biology of the Cell 16, no. 12 (December 2005): 5819–31. http://dx.doi.org/10.1091/mbc.e05-07-0685.
Full textAbstract:
In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.
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Zachar, Z., J. Kramer, I. P. Mims, and P. M. Bingham. "Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans." Journal of Cell Biology 121, no. 4 (May 15, 1993): 729–42. http://dx.doi.org/10.1083/jcb.121.4.729.
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We report studies using an enhanced experimental system to investigate organization of nuclear pre-mRNA metabolism. It is based on the powerful genetic system and polytene nuclei of Drosophila. We observe (at steady state) movement of a specific pre-mRNA between its gene and the nuclear surface. This movement is isotropic, at rates consistent with diffusion and is restricted to a small nuclear subcompartment defined by exclusion from chromosome axes and the nucleolus. Bulk polyadenylated nuclear pre-mRNA precisely localizes in this same subcompartment indicating that most or all pre-mRNAs use the same route of intranuclear movement. In addition to association with nascent transcripts, snRNPs are coconcentrated with pre-mRNA in this subcompartment. In contrast to constitutive splices, at least one regulated splice occurs slowly and may undergo execution remotely from the site of pre-mRNA synthesis. Details of our results suggest that retention of incompletely spliced pre-mRNA is a function of the nuclear surface. We propose a simple model--based on channeled diffusion--for organization of intranuclear transport and metabolism of pre-mRNAs in polytene nuclei. We argue that this model can be generalized to all metazoan nuclei.
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Weyrich, Andrew S., Larry W. Kraiss, and Guy A. Zimmerman. "Trading places: mRNA transfer between cells." Blood 110, no. 7 (October 1, 2007): 2219. http://dx.doi.org/10.1182/blood-2007-06-093740.
Full textAbstract:
Cells use receptor-ligand interactions and endocrine, paracrine, and juxtacrine mechanisms to transmit signals to one another. Deregibus and colleagues now report that information transfer between cells also occurs through horizontal transfer of mRNA-rich microvesicles (MVs).
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Sakharov, Pavel A., Egor A. Smolin, Dmitry N. Lyabin, and Sultan C. Agalarov. "ATP-Independent Initiation during Cap-Independent Translation of m6A-Modified mRNA." International Journal of Molecular Sciences 22, no. 7 (April 1, 2021): 3662. http://dx.doi.org/10.3390/ijms22073662.
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The methylation of adenosine in the N6 position (m6A) is a widely used modification of eukaryotic mRNAs. Its importance for the regulation of mRNA translation was put forward recently, essentially due to the ability of methylated mRNA to be translated in conditions of inhibited cap-dependent translation initiation, e.g., under stress. However, the peculiarities of translation initiation on m6A-modified mRNAs are not fully known. In this study, we used toeprinting and translation in a cell-free system to confirm that m6A-modified mRNAs can be translated in conditions of suppressed cap-dependent translation. We show for the first time that m6A-modified mRNAs display not only decreased elongation, but also a lower efficiency of translation initiation. Additionally, we report relative resistance of m6A-mRNA translation initiation in the absence of ATP and inhibited eIF4A activity. Our novel findings indicate that the scanning of m6A-modified leader sequences is performed by a noncanonical mechanism.
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Bohjanen, P. R., B. Petryniak, C. H. June, C. B. Thompson, and T. Lindsten. "An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA." Molecular and Cellular Biology 11, no. 6 (June 1991): 3288–95. http://dx.doi.org/10.1128/mcb.11.6.3288.
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Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.
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Bohjanen, P. R., B. Petryniak, C. H. June, C. B. Thompson, and T. Lindsten. "An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA." Molecular and Cellular Biology 11, no. 6 (June 1991): 3288–95. http://dx.doi.org/10.1128/mcb.11.6.3288-3295.1991.
Full textAbstract:
Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.
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Goodwin, R. G., F. M. Rottman, T. Callaghan, H. J. Kung, P. A. Maroney, and T. W. Nilsen. "c-erbB activation in avian leukosis virus-induced erythroblastosis: multiple epidermal growth factor receptor mRNAs are generated by alternative RNA processing." Molecular and Cellular Biology 6, no. 9 (September 1986): 3128–33. http://dx.doi.org/10.1128/mcb.6.9.3128.
Full textAbstract:
Avian leukosis virus-induced erythroblastosis results from the specific interruption of the host oncogene, c-erbB, by the insertion of an intact provirus. This insertion results in the expression of two size classes (3.6 and 7.0 kilobases [kb]) of truncated c-erbB transcripts which are initiated in the 5' long terminal repeat of the integrated provirus. Through sequence analysis of erbB cDNA clones we have previously shown that the 3.6-kb activated erbB mRNA contains portions of viral gag and env genes fused to c-erbB sequences (T.W. Nilsen, P.A. Maroney, R.G. Goodwin, F.M. Rottman, L.B. Crittenden, M.A. Raines, and H.-J. Kung, Cell 41:719-726, 1985). In this report we show that the 7-kb mRNA differs from the shorter activated c-erbB mRNA in the length of its 3' untranslated sequence such that the longer mRNA has an extremely long (4.3 kb) 3' untranslated sequence. Additionally, we demonstrate that activated c-erbB mRNA precursors can be processed by alternative splicing to yield mRNAs with viral gag sequences fused directly to c-erbB sequences. Finally, blot hybridization evidence suggests that the two size classes of activated c-erbB mRNA in erythroblastic tissue represent truncated versions of the two c-erbB mRNAs present in normal tissue.
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Surdej, P., and M. Jacobs-Lorena. "Developmental Regulation of bicoid mRNA Stability Is Mediated by the First 43 Nucleotides of the 3′ Untranslated Region." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2892–900. http://dx.doi.org/10.1128/mcb.18.5.2892.
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ABSTRACT During the transition from the maternal to the zygotic developmental program, the expression of genes important for pattern formation or cell cycle regulation changes dramatically. Rapid changes in gene expression are achieved in part through the control of mRNA stability. This report focuses on bicoid, a gene essential for formation of anterior embryonic structures in Drosophila melanogaster. bicoid mRNA is synthesized exclusively during oogenesis. Here, we show that bicoid mRNA stability is regulated. While bicoid mRNA is stable in retained oocytes, in unfertilized eggs, and during the first 2 h of embryogenesis, specific degradation is activated at cellularization of the blastoderm. To identify cis-acting sequences required for bicoid mRNA’s regulated stability, fusions betweenbicoid and genes producing stable mRNAs were introduced into the Drosophila germ line by P-element-mediated transformation. The analysis of the fusion mRNAs identified abicoid instability element (BIE) contained within a 43-nucleotide sequence immediately following the stop codon. The BIE is sufficient to destabilize the otherwise-stable ribosomal protein A1 mRNA and is separable from the previously identified bicoidmRNA localization signals and from the “nanos response element.” Similar mechanisms may regulate a class of developmentally important maternal genes whose mRNA has a temporal profile similar to that of bicoid.
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Petersen, R. B., and S. Lindquist. "Regulation of HSP70 synthesis by messenger RNA degradation." Cell Regulation 1, no. 1 (November 1989): 135–49. http://dx.doi.org/10.1091/mbc.1.1.135.
Full textAbstract:
When Drosophila cells are heat shocked, hsp70 messenger RNA (mRNA) is stable and is translated at high efficiencies. During recovery from heat shock, hsp70 synthesis is repressed and its messenger RNA (mRNA) is degraded in a highly regulated fashion. Dramatic differences in the timing of repression and degradation are observed after heat treatments of different severities. The 3' untranslated region (UTR) of the hsp70 mRNA was sufficient to transfer this regulated degradation to heterologous mRNAs. Altering the translational efficiency of the message or changing its natural translation-termination site did not alter its pattern of regulation, although in some cases it changed the absolute rate of degradation. We have previously shown that hsp70 mRNA is very unstable when it is expressed at normal growth temperatures (from a metallothionein promoter). We report here that the 3' untranslated region of the hsp70 mRNA is responsible for this instability as well. We postulate that a mechanism for degrading hsp70 mRNA pre-exists in Drosophila cells, that it is inactivated by heat shock and that it is the reactivation of this mechanism that is responsible for hsp70 repression during recovery. This degradation system may be the same as that used by other unstable mRNAs.
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Goodwin, R. G., F. M. Rottman, T. Callaghan, H. J. Kung, P. A. Maroney, and T. W. Nilsen. "c-erbB activation in avian leukosis virus-induced erythroblastosis: multiple epidermal growth factor receptor mRNAs are generated by alternative RNA processing." Molecular and Cellular Biology 6, no. 9 (September 1986): 3128–33. http://dx.doi.org/10.1128/mcb.6.9.3128-3133.1986.
Full textAbstract:
Avian leukosis virus-induced erythroblastosis results from the specific interruption of the host oncogene, c-erbB, by the insertion of an intact provirus. This insertion results in the expression of two size classes (3.6 and 7.0 kilobases [kb]) of truncated c-erbB transcripts which are initiated in the 5' long terminal repeat of the integrated provirus. Through sequence analysis of erbB cDNA clones we have previously shown that the 3.6-kb activated erbB mRNA contains portions of viral gag and env genes fused to c-erbB sequences (T.W. Nilsen, P.A. Maroney, R.G. Goodwin, F.M. Rottman, L.B. Crittenden, M.A. Raines, and H.-J. Kung, Cell 41:719-726, 1985). In this report we show that the 7-kb mRNA differs from the shorter activated c-erbB mRNA in the length of its 3' untranslated sequence such that the longer mRNA has an extremely long (4.3 kb) 3' untranslated sequence. Additionally, we demonstrate that activated c-erbB mRNA precursors can be processed by alternative splicing to yield mRNAs with viral gag sequences fused directly to c-erbB sequences. Finally, blot hybridization evidence suggests that the two size classes of activated c-erbB mRNA in erythroblastic tissue represent truncated versions of the two c-erbB mRNAs present in normal tissue.
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Castagnola, P., B. Dozin, G. Moro, and R. Cancedda. "Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro." Journal of Cell Biology 106, no. 2 (February 1, 1988): 461–67. http://dx.doi.org/10.1083/jcb.106.2.461.
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This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary, types II and IX collagen mRNAs sharply increased within the first week of suspension culture, peaked in the second week, and thereafter began to decrease. This decrease was particularly significant for type IX collagen mRNA. The level of type X collagen mRNA progressively increased during the course of the culture, reached its maximal value after 3-4 wk, and decreased only at a later stage of cell differentiation. As determined by in vitro run-off transcription assays, all these changes in collagen mRNA levels could be attributed to parallel modifications in the relative rate of transcription of the corresponding collagen genes. We suggest that chicken chondrocyte differentiation proceeds through at least two different steps: (a) first, transition from a stage characterized by a high level of type I collagen mRNA to a stage characterized by predominance of types II and IX collagen mRNAs; (b) later, transition to a stage characterized by the highest level of type X collagen mRNA.
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Liao, Wan-Lin, Wei-Chiao Wang, Wen-Chang Chang, and Joseph T. Tseng. "The RNA-binding Protein HuR Stabilizes Cytosolic Phospholipase A2α mRNA under Interleukin-1β Treatment in Non-small Cell Lung Cancer A549 Cells." Journal of Biological Chemistry 286, no. 41 (August 23, 2011): 35499–508. http://dx.doi.org/10.1074/jbc.m111.263582.
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The activation of cytosolic phospholipase A2α (cPLA2α) plays an important role in initiating the inflammatory response. The regulation of cPLA2α mRNA turnover has been proposed to control cPLA2α gene expression under cytokine and growth factor stimulation. However, the detailed mechanism is still unknown. In this report, we have demonstrated that the cPLA2α mRNA stability was increased under IL-1β treatment in A549 cells. By using EMSAs, HuR was identified as binding with the cPLA2α mRNA 3′-UTR, and the binding region was located at nucleotides 2716–2807, a fragment containing AUUUA flanked by U-rich sequences. IL-1β treatment enhanced the association of cPLA2α mRNA with cytosolic HuR. The reduction of HuR expression by RNA interference technology inhibited IL-1β-induced cPLA2α mRNA and protein expression. Furthermore, blocking the p38 MAPK signaling pathway with SB203580 abolished the effect of IL-1β-induced cPLA2α gene expression. Phosphorylation at residue Thr-118 of HuR is crucial in regulating the interaction between HuR and its target mRNAs. Mutation of HuR Thr-118 reduced the association between HuR and cPLA2α mRNA under IL-1β treatment. This inhibitory effect was also observed in binding with COX-2 mRNA. This result indicated that p38 MAPK-mediated Thr-118 phosphorylation may play a key role in regulating the interaction of HuR with its target mRNAs in inflammation.
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Banihashemi, Lili, Gerald M. Wilson, Neha Das, and Gary Brewer. "Upf1/Upf2 Regulation of 3′ Untranslated Region Splice Variants of AUF1 Links Nonsense-Mediated and A+U-Rich Element-Mediated mRNA Decay." Molecular and Cellular Biology 26, no. 23 (September 25, 2006): 8743–54. http://dx.doi.org/10.1128/mcb.02251-05.
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ABSTRACT AUF1 is an RNA-binding protein that targets mRNAs containing A+U-rich elements (AREs) for rapid cytoplasmic turnover. Alternative pre-mRNA splicing produces five variants of AUF1 mRNA that differ in the composition of their 3′-untranslated regions (3′-UTRs). Previous work suggested that this heterogeneity in 3′-UTR sequence could regulate AUF1 expression by two potential mechanisms. First, AUF1 may regulate its own expression by binding to AREs in 3′-UTR splice variants that retain intron 9. The second potential mechanism, and the focus of this report, is regulation of a subset of 3′-UTR splice variants by the nonsense-mediated mRNA decay (NMD) pathway. Two of the five AUF1 mRNA 3′-UTR variants position the translational termination codon more than 50 nucleotides upstream of an exon-exon junction, creating a potential triggering signal for NMD in mammalian cells. Disruption of cellular NMD pathways by RNA interference-mediated knockdown of Upf1/Rent1 or Upf2/Rent2 or transfection of a dominant-negative Upf1 mutant specifically enhanced expression of these two candidate NMD substrate mRNAs in cells, involving stabilization of each transcript. Ribonucleoprotein immunoprecipitation experiments revealed that both Upf1 and Upf2 can associate with an NMD-sensitive AUF1 mRNA 3′-UTR variant in cells. Finally, quantitation of AUF1 mRNA 3′-UTR splice variants during murine embryonic development showed that the expression of NMD-sensitive AUF1 mRNAs is specifically enhanced as development proceeds, contributing to dynamic changes in AUF1 3′-UTR structures during embryogenesis. Together, these studies provide the first evidence of linkage between the nonsense- and ARE-mediated mRNA decay pathways, which may constitute a new mechanism regulating the expression of ARE-containing mRNAs.
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Mettel, Carsten, Yongkyu Kim, Pravin Malla Shrestha, and Werner Liesack. "Extraction of mRNA from Soil." Applied and Environmental Microbiology 76, no. 17 (July 9, 2010): 5995–6000. http://dx.doi.org/10.1128/aem.03047-09.
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ABSTRACT Here, we report an efficient method for extracting high-quality mRNA from soil. Key steps in the isolation of total RNA were low-pH extraction (pH 5.0) and Q-Sepharose chromatography. The removal efficiency of humic acids was 94 to 98% for all soils tested. To enrich mRNA, subtractive hybridization of rRNA was most efficient. Subtractive hybridization may be followed by exonuclease treatment if the focus is on the analysis of unprocessed mRNA. The total extraction method can be completed within 8 h, resulting in enriched mRNA ranging from 200 bp to 4 kb in size.
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Liang, Xue-hai, Joshua G. Nichols, Cheryl L. De Hoyos, and Stanley T. Crooke. "Some ASOs that bind in the coding region of mRNAs and induce RNase H1 cleavage can cause increases in the pre-mRNAs that may blunt total activity." Nucleic Acids Research 48, no. 17 (September 1, 2020): 9840–58. http://dx.doi.org/10.1093/nar/gkaa715.
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Abstract Antisense oligonucleotide (ASO) drugs that trigger RNase H1 cleavage of target RNAs have been developed to treat various diseases. Basic pharmacological principles suggest that the development of tolerance is a common response to pharmacological interventions. In this manuscript, for the first time we report a molecular mechanism of tolerance that occurs with some ASOs. Two observations stimulated our interest: some RNA targets are difficult to reduce with RNase H1 activating ASOs and some ASOs display a shorter duration of activity than the prolonged target reduction typically observed. We found that certain ASOs targeting the coding region of some mRNAs that initially reduce target mRNAs can surprisingly increase the levels of the corresponding pre-mRNAs. The increase in pre-mRNA is delayed and due to enhanced transcription and likely also slower processing. This process requires that the ASOs bind in the coding region and reduce the target mRNA by RNase H1 while the mRNA resides in the ribosomes. The pre-mRNA increase is dependent on UPF3A and independent of the NMD pathway or the XRN1-CNOT pathway. The response is consistent in multiple cell lines and independent of the methods used to introduce ASOs into cells.
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Rao, T. R., and L. I. Slobin. "The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate." Molecular and Cellular Biology 8, no. 3 (March 1988): 1085–92. http://dx.doi.org/10.1128/mcb.8.3.1085.
Full textAbstract:
The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.
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Rao, T. R., and L. I. Slobin. "The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate." Molecular and Cellular Biology 8, no. 3 (March 1988): 1085–92. http://dx.doi.org/10.1128/mcb.8.3.1085-1092.1988.
Full textAbstract:
The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.
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Child, Jessica R., Qiang Chen, David W. Reid, Sujatha Jagannathan, and Christopher V. Nicchitta. "Recruitment of endoplasmic reticulum-targeted and cytosolic mRNAs into membrane-associated stress granules." RNA 27, no. 10 (July 8, 2021): 1241–56. http://dx.doi.org/10.1261/rna.078858.121.
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Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.
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Sikorski, Pawel J., Marcin Warminski, Dorota Kubacka, Tomasz Ratajczak, Dominika Nowis, Joanna Kowalska, and Jacek Jemielity. "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells." Nucleic Acids Research 48, no. 4 (January 27, 2020): 1607–26. http://dx.doi.org/10.1093/nar/gkaa032.
Full textAbstract:
Abstract 7-Methylguanosine 5′ cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5′-terminal nucleotides and additional methylations (2′-O-methylation and m6A). Currently available 5′-capping methods do not address this diversity. We report trinucleotide 5′ cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2′-O-methylation). HPLC-purified mRNAs carrying these 5′ caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5′-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2′-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.
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Perrot-Rechenmann, C., M. Joannes, D. Squalli, and P. Lebacq. "Detection of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylase transcripts in maize leaves by in situ hybridization with sulfonated cDNA probes." Journal of Histochemistry & Cytochemistry 37, no. 4 (April 1989): 423–28. http://dx.doi.org/10.1177/37.4.2926120.
Full textAbstract:
This report outlines an efficient in situ hybridization method for locating specific mRNAs in tissue cryosections using sulfonated cDNA probes. The method involves chemical modification of DNA probes by insertion of a sulfone radical on cytosine residues, which generates a specific epitope. Sulfonated DNA is then detected by using indirect immunochemical procedure. Alternatively, antibodies conjugated to fluorescein or to alkaline phosphatase were used for mRNA detection. In situ hybridization was developed to study aspects of mesophyll and bundle sheath cell differentiation in maize leaves. Our results indicate that phosphoenolpyruvate carboxylase (PEP C) mRNA is restricted to mesophyll cells, and the nucleus-encoded mRNA of the small subunit (SSU) ribulose 1,5-bisphosphate carboxylase (RuBP C) is limited to the cytosol of bundle sheath cells. Thus, using in situ hybridization, we have demonstrated that the differential distribution of PEP C and RuBP C proteins in the two cell types also reflects the location of their mRNAs. These data imply either a tissue-specific transcriptional regulation or a selective mRNA degradation.
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Bardwell, V. J., D. Zarkower, M. Edmonds, and M. Wickens. "The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase." Molecular and Cellular Biology 10, no. 2 (February 1990): 846–49. http://dx.doi.org/10.1128/mcb.10.2.846.
Full textAbstract:
Virtually all mRNAs in eucaryotes end in a poly(A) tail. This tail is added posttranscriptionally. In this report, we demonstrate that the enzyme that catalyzes this modification is identical with an activity first identified 30 years ago, the function of which was previously unknown. This enzyme, poly(A) polymerase, lacks any intrinsic specificity for its mRNA substrate but gains specificity by interacting with distinct molecules: a poly(A) polymerase from calf thymus, when combined with specificity factor(s) from cultured human cells, specifically and efficiently polyadenylates only appropriate mRNA substrates. Our results thus demonstrate that this polymerase is responsible for the addition of poly(A) to mRNAs and that its interaction with specificity factors is conserved.
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Bardwell, V. J., D. Zarkower, M. Edmonds, and M. Wickens. "The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase." Molecular and Cellular Biology 10, no. 2 (February 1990): 846–49. http://dx.doi.org/10.1128/mcb.10.2.846-849.1990.
Full textAbstract:
Virtually all mRNAs in eucaryotes end in a poly(A) tail. This tail is added posttranscriptionally. In this report, we demonstrate that the enzyme that catalyzes this modification is identical with an activity first identified 30 years ago, the function of which was previously unknown. This enzyme, poly(A) polymerase, lacks any intrinsic specificity for its mRNA substrate but gains specificity by interacting with distinct molecules: a poly(A) polymerase from calf thymus, when combined with specificity factor(s) from cultured human cells, specifically and efficiently polyadenylates only appropriate mRNA substrates. Our results thus demonstrate that this polymerase is responsible for the addition of poly(A) to mRNAs and that its interaction with specificity factors is conserved.
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Jefferson, Leonard S., and Scot R. Kimball. "Translational Control of Protein Synthesis: Implications for Understanding Changes in Skeletal Muscle Mass." International Journal of Sport Nutrition and Exercise Metabolism 11, s1 (December 2001): S143—S149. http://dx.doi.org/10.1123/ijsnem.11.s1.s143.
Full textAbstract:
Gain or loss of skeletal muscle mass is due largely to the establishment of an imbalance between rates of protein synthesis and degradation. A key determinant of the rate of protein synthesis is translation initiation, a process regulated in part through binding of initiator methionyl-tRNA (met-tRNAi) and messenger RNA (mRNA) to a 40S ribosomal subunit. Either the met-tRNAi or mRNA binding step can become limiting for protein synthesis. Furthermore, the mRNA binding step can modulate translation of specific mRNAs with or without changes in the overall rate of protein synthesis. This report highlights molecular mechanisms involved in mediating control of the mRNA binding step in translation initiation. Particular attention is given to the effect of exercise on this step and to how the branched-chain amino acid leucine stimulates muscle protein synthesis after exercise. Potential mechanisms for exercise induced increase in muscle mass are discussed.
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Cragle, Chad E., Melanie C. MacNicol, Stephanie D. Byrum, Linda L. Hardy, Samuel G. Mackintosh, William A. Richardson, Nicola K. Gray, Gwen V. Childs, Alan J. Tackett, and Angus M. MacNicol. "Musashi interaction with poly(A)-binding protein is required for activation of target mRNA translation." Journal of Biological Chemistry 294, no. 28 (May 31, 2019): 10969–86. http://dx.doi.org/10.1074/jbc.ra119.007220.
Full textAbstract:
The Musashi family of mRNA translational regulators controls both physiological and pathological stem cell self-renewal primarily by repressing target mRNAs that promote differentiation. In response to differentiation cues, Musashi can switch from a repressor to an activator of target mRNA translation. However, the molecular events that distinguish Musashi-mediated translational activation from repression are not understood. We have previously reported that Musashi function is required for the maturation of Xenopus oocytes and specifically for translational activation of specific dormant maternal mRNAs. Here, we employed MS to identify cellular factors necessary for Musashi-dependent mRNA translational activation. We report that Musashi1 needs to associate with the embryonic poly(A)-binding protein (ePABP) or the canonical somatic cell poly(A)-binding protein PABPC1 for activation of Musashi target mRNA translation. Co-immunoprecipitation studies demonstrated an increased Musashi1 interaction with ePABP during oocyte maturation. Attenuation of endogenous ePABP activity severely compromised Musashi function, preventing downstream signaling and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP-attenuated oocytes. Consistent with these Xenopus findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Because association of Musashi1 with poly(A)-binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly(A)-binding protein family members in response to extracellular cues that control cell fate.