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Journal articles on the topic 'MRNA'
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1
Chiang, Chiayn, Guang-Wu Chen, and Shin-Ru Shih. "Mutations at Alternative 5′ Splice Sites of M1 mRNA Negatively Affect Influenza A Virus Viability and Growth Rate." Journal of Virology 82, no. 21 (September 3, 2008): 10873–86. http://dx.doi.org/10.1128/jvi.00506-08.
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ABSTRACT Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5′ splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5′ splice site), C51G (at the 3′ end of the exon of the M2 mRNA 5′ splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3′ end of exon of the mRNA3 5′ splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3′ end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5′ splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.
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Jackson, David, and Robert A. Lamb. "The influenza A virus spliced messenger RNA M mRNA3 is not required for viral replication in tissue culture." Journal of General Virology 89, no. 12 (December 1, 2008): 3097–101. http://dx.doi.org/10.1099/vir.0.2008/004739-0.
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Influenza A virus genome RNA segment 7 encodes three known mRNAs, two of which, M2 mRNA and M mRNA3, are derived by alternative splicing of the primary collinear mRNA transcript using alternative 5′ splice sites. The function of M mRNA3 is currently unknown, therefore we attempted to determine whether it is essential for virus replication. Recombinant viruses unable to produce M mRNA3 and/or M2 mRNA were created by mutating the shared 3′ splice site. Growth of the mutant viruses in M2-expressing MDCK cells was not significantly affected by the lack of M mRNA3. During the course of a wild-type virus infection, levels of M mRNA3 began to decrease while those of M2 mRNA increased, which may indicate a potential mechanism of alternative splicing control. These data suggest that neither M mRNA3 nor any potential protein product are essential for influenza virus replication in tissue culture.
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Ruepp, Marc-David, Chiara Aringhieri, Silvia Vivarelli, Stefano Cardinale, Simona Paro, Daniel Schümperli, and Silvia M. L. Barabino. "Mammalian pre-mRNA 3′ End Processing Factor CF Im68 Functions in mRNA Export." Molecular Biology of the Cell 20, no. 24 (December 15, 2009): 5211–23. http://dx.doi.org/10.1091/mbc.e09-05-0389.
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Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3′ end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3′ end processing complex, CF Im68, stimulates mRNA export. CF Im68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF Im68 may act as a novel adaptor for NXF1/TAP, we show that CF Im68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF Im68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3′ end processing factor CF Im68 in mRNA export.
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Noble, Scott L., Brittany L. Allen, Lai Kuan Goh, Kristen Nordick, and Thomas C. Evans. "Maternal mRNAs are regulated by diverse P body–related mRNP granules during early Caenorhabditis elegans development." Journal of Cell Biology 182, no. 3 (August 11, 2008): 559–72. http://dx.doi.org/10.1083/jcb.200802128.
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Processing bodies (P bodies) are conserved mRNA–protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body–like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3′ untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development.
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Yu, Jia, and J. Eric Russell. "Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA." Molecular and Cellular Biology 21, no. 17 (September 1, 2001): 5879–88. http://dx.doi.org/10.1128/mcb.21.17.5879-5888.2001.
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ABSTRACT Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.
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Robb, Nicole C., and Ervin Fodor. "The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein." Journal of General Virology 93, no. 1 (January 1, 2012): 113–18. http://dx.doi.org/10.1099/vir.0.035485-0.
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The influenza A virus M1 mRNA is alternatively spliced to produce M2 mRNA, mRNA3, and in some cases, M4 mRNA. Splicing of influenza mRNAs is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mRNAs encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mRNAs in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mRNAs compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mRNA.
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Schmidt, Edward E., Eric S. Hanson, and Mario R. Capecchi. "Sequence-Independent Assembly of Spermatid mRNAs into Messenger Ribonucleoprotein Particles." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3904–15. http://dx.doi.org/10.1128/mcb.19.5.3904.
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ABSTRACT During mammalian spermatogenesis, meiosis is followed by a brief period of high transcriptional activity. At this time a large amount of mRNA is stored as messenger ribonucleoprotein (mRNP) particles. All subsequent processes of sperm maturation occur in the complete absence of transcription, primarily using proteins which are newly synthesized from these stored mRNAs. By expressing transgene mRNAs in the early haploid spermatids of mice, we have investigated the sequence requirements for determining whether specific mRNAs in these cells will be stored as mRNP particles or be assembled into polysomes. The results suggest that mRNAs which are transcribed in spermatids are assembled into mRNP particles by a mechanism that acts independently of mRNA sequence. Our findings reveal a fundamental similarity between the mechanisms of translational control used in spermatogenesis and oogenesis.
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Kleene, Kenneth C., and Danielle L. Cullinane. "Maybe repressed mRNAs are not stored in the chromatoid body in mammalian spermatids." REPRODUCTION 142, no. 3 (September 2011): 383–88. http://dx.doi.org/10.1530/rep-11-0113.
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The chromatoid body is a dynamic organelle that is thought to coordinate the cytoplasmic regulation of mRNA translation and degradation in mammalian spermatids. The chromatoid body is also postulated to function in repression of mRNA translation by sequestering dormant mRNAs where they are inaccessible to the translational apparatus. This review finds no convincing evidence that dormant mRNAs are localized exclusively in the chromatoid body. This discrepancy can be explained by two hypotheses. First, experimental artifacts, possibly related to peculiarities of the structure and function of the chromatoid body, preclude obtaining an accurate indication of mRNA localization. Second, mRNA is not stored in the chromatoid body, because, like perinuclear P granules in Caenorhabditis elegans, the chromatoid body functions as a center for mRNP remodeling and export to other cytoplasmic sites.
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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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Yang, Feng, Yong Peng, Elizabeth L. Murray, Yuichi Otsuka, Nancy Kedersha, and Daniel R. Schoenberg. "Polysome-Bound Endonuclease PMR1 Is Targeted to Stress Granules via Stress-Specific Binding to TIA-1." Molecular and Cellular Biology 26, no. 23 (September 18, 2006): 8803–13. http://dx.doi.org/10.1128/mcb.00090-06.
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ABSTRACT The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the α subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional ∼680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.
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Zarnack, Kathi, and Michael Feldbrügge. "Microtubule-Dependent mRNA Transport in Fungi." Eukaryotic Cell 9, no. 7 (May 14, 2010): 982–90. http://dx.doi.org/10.1128/ec.00030-10.
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ABSTRACT The localization and local translation of mRNAs constitute an important mechanism to promote the correct subcellular targeting of proteins. mRNA localization is mediated by the active transport of mRNPs, large assemblies consisting of mRNAs and associated factors such as RNA-binding proteins. Molecular motors move mRNPs along the actin or microtubule cytoskeleton for short-distance or long-distance trafficking, respectively. In filamentous fungi, microtubule-based long-distance transport of vesicles, which are involved in membrane and cell wall expansion, supports efficient hyphal growth. Recently, we discovered that the microtubule-mediated transport of mRNAs is essential for the fast polar growth of infectious filaments in the corn pathogen Ustilago maydis. Combining in vivo UV cross-linking and RNA live imaging revealed that the RNA-binding protein Rrm4, which constitutes an integral part of the mRNP transport machinery, mediates the transport of distinct mRNAs encoding polarity factors, protein synthesis factors, and mitochondrial proteins. Moreover, our results indicate that microtubule-dependent mRNA transport is evolutionarily conserved from fungi to higher eukaryotes. This raises the exciting possibility of U. maydis as a model system to uncover basic concepts of long-distance mRNA transport.
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Bier, Katja, Ashley York, and Ervin Fodor. "Cellular cap-binding proteins associate with influenza virus mRNAs." Journal of General Virology 92, no. 7 (July 1, 2011): 1627–34. http://dx.doi.org/10.1099/vir.0.029231-0.
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The influenza virus RNA polymerase synthesizes three types of RNA: genomic vRNA, anti-genomic cRNA and mRNA. Both vRNA and cRNA are bound by the viral RNA polymerase and nucleoprotein to form ribonucleoprotein complexes. Viral mRNAs are also proposed to be bound by the RNA polymerase to prevent their endonucleolytic cleavage, regulate the splicing of M1 mRNA, and facilitate translation. Here, we used standard immunoprecipitation, biochemical purification and RNA immunoprecipitation assays to investigate the association of viral and host factors with viral mRNA. We found that viral mRNA associates with the viral non-structural protein 1 (NS1), cellular poly(A)-binding protein 1 (PABP1), the 20 kDa subunit NCBP1 of the nuclear cap-binding complex (CBC), the RNA and export factor-binding protein REF/Aly and the translation initiation factor eIF4E. However, our data suggest that the RNA polymerase might not form part of the viral messenger ribonucleoprotein (mRNP) complex. We propose a model in which viral mRNAs, by associating with cellular cap-binding proteins, follow the pathways normally used by cellular mRNAs for splicing, nuclear export and translation.
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Berger, Lloyd C., Jnanankur Bag, and Bruce H. Sells. "Translation of poly(A)-binding protein mRNA is regulated by growth conditions." Biochemistry and Cell Biology 70, no. 9 (September 1, 1992): 770–78. http://dx.doi.org/10.1139/o92-117.
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Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (~ 60%) and resting cells (~ 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5′ untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.Key words: translation, mRNP, poly(A) binding protein.
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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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Saroufim, Mark-Albert, Pierre Bensidoun, Pascal Raymond, Samir Rahman, Matthew R. Krause, Marlene Oeffinger, and Daniel Zenklusen. "The nuclear basket mediates perinuclear mRNA scanning in budding yeast." Journal of Cell Biology 211, no. 6 (December 21, 2015): 1131–40. http://dx.doi.org/10.1083/jcb.201503070.
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After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.
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Minich, Waldemar B., Elena V. Volyanik, Nadezhda L. Korneyeva, Yuri V. Berezin, and Lev P. Ovchinnikov. "Cytoplasmic mRNP proteins affect mRNA translation." Molecular Biology Reports 14, no. 2-3 (1990): 65–67. http://dx.doi.org/10.1007/bf00360418.
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Windgassen, Merle, Dorothée Sturm, Iván J. Cajigas, Carlos I. González, Matthias Seedorf, Holger Bastians, and Heike Krebber. "Yeast Shuttling SR Proteins Npl3p, Gbp2p, and Hrb1p Are Part of the Translating mRNPs, and Npl3p Can Function as a Translational Repressor." Molecular and Cellular Biology 24, no. 23 (December 1, 2004): 10479–91. http://dx.doi.org/10.1128/mcb.24.23.10479-10491.2004.
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ABSTRACT A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.
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Stöhr, Nadine, Marcell Lederer, Claudia Reinke, Sylke Meyer, Mechthild Hatzfeld, Robert H. Singer, and Stefan Hüttelmaier. "ZBP1 regulates mRNA stability during cellular stress." Journal of Cell Biology 175, no. 4 (November 13, 2006): 527–34. http://dx.doi.org/10.1083/jcb.200608071.
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An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein–mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.
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Schlautmann, Lena P., and Niels H. Gehring. "A Day in the Life of the Exon Junction Complex." Biomolecules 10, no. 6 (June 5, 2020): 866. http://dx.doi.org/10.3390/biom10060866.
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The exon junction complex (EJC) is an abundant messenger ribonucleoprotein (mRNP) component that is assembled during splicing and binds to mRNAs upstream of exon-exon junctions. EJCs accompany the mRNA during its entire life in the nucleus and the cytoplasm and communicate the information about the splicing process and the position of introns. Specifically, the EJC’s core components and its associated proteins regulate different steps of gene expression, including pre-mRNA splicing, mRNA export, translation, and nonsense-mediated mRNA decay (NMD). This review summarizes the most important functions and main protagonists in the life of the EJC. It also provides an overview of the latest findings on the assembly, composition and molecular activities of the EJC and presents them in the chronological order, in which they play a role in the EJC’s life cycle.
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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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Darbouy, M., M. N. Chobert, O. Lahuna, T. Okamoto, J. P. Bonvalet, N. Farman, and Y. Laperche. "Tissue-specific expression of multiple gamma-glutamyl transpeptidase mRNAs in rat epithelia." American Journal of Physiology-Cell Physiology 261, no. 6 (December 1, 1991): C1130—C1137. http://dx.doi.org/10.1152/ajpcell.1991.261.6.c1130.
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gamma-Glutamyl transpeptidase (GGT) is an enzyme that plays a key role in interorgan glutathione transport. Three mRNAs (mRNAI, mRNAII, and mRNAIII) are known to encode the GGT precursor; they are initiated on three separate promoters on the single GGT gene. In this work, we identified by Northern blot and RNase H analysis a new GGT mRNA (mRNAIV). This mRNA differs from the others in its 5'-noncoding sequence. This mRNA species is the predominant GGT mRNA expressed in HTC hepatoma cells and in the small intestine in which its level increases from the base to the apex of the microvillus. The analysis of the GGT gene expression pattern in kidney, mammary gland, small intestine, liver, preneoplastic liver, and HTC hepatoma cells reveals a strong tissue or cell specificity. The mRNAIII was found in all the tissues and cells; in contrast, the expression of mRNAI, mRNAII, and mRNAIV is limited in normal tissues to the kidney and to the small intestine, the two tissues that display the highest enzyme activity. The synthesis of these three mRNAs is linked to the development of the kidney proximal tubule and to the differentiation of the enterocyte. The tissue and cell specificity of the GGT gene expression is based upon the use of multiple promoters that are controlled independently by specific cell factors.
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Zheng, Dinghai, Nader Ezzeddine, Chyi-Ying A. Chen, Wenmiao Zhu, Xiangwei He, and Ann-Bin Shyu. "Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells." Journal of Cell Biology 182, no. 1 (July 14, 2008): 89–101. http://dx.doi.org/10.1083/jcb.200801196.
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Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5′-to-3′ degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.
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Brengues, Muriel, and Roy Parker. "Accumulation of Polyadenylated mRNA, Pab1p, eIF4E, and eIF4G with P-Bodies inSaccharomyces cerevisiae." Molecular Biology of the Cell 18, no. 7 (July 2007): 2592–602. http://dx.doi.org/10.1091/mbc.e06-12-1149.
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Recent experiments have shown that mRNAs can move between polysomes and P-bodies, which are aggregates of nontranslating mRNAs associated with translational repressors and the mRNA decapping machinery. The transitions between polysomes and P-bodies and how the poly(A) tail and the associated poly(A) binding protein 1 (Pab1p) may affect this process are unknown. Herein, we provide evidence that poly(A)+mRNAs can enter P-bodies in yeast. First, we show that both poly(A)−and poly(A)+mRNA become translationally repressed during glucose deprivation, where mRNAs accumulate in P-bodies. In addition, both poly(A)+transcripts and/or Pab1p can be detected in P-bodies during glucose deprivation and in stationary phase. Cells lacking Pab1p have enlarged P-bodies, suggesting that Pab1p plays a direct or indirect role in shifting the equilibrium of mRNAs away from P-bodies and into translation, perhaps by aiding in the assembly of a type of mRNP within P-bodies that is poised to reenter translation. Consistent with this latter possibility, we observed the translation initiation factors (eIF)4E and eIF4G in P-bodies at a low level during glucose deprivation and at high levels in stationary phase. Moreover, Pab1p exited P-bodies much faster than Dcp2p when stationary phase cells were given fresh nutrients. Together, these results suggest that polyadenylated mRNAs can enter P-bodies, and an mRNP complex including poly(A)+mRNA, Pab1p, eIF4E, and eIF4G2 may represent a transition state during the process of mRNAs exchanging between P-bodies and translation.
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Walters, Robert, and Roy Parker. "Is there quality control of localized mRNAs?" Journal of Cell Biology 204, no. 6 (March 17, 2014): 863–68. http://dx.doi.org/10.1083/jcb.201401059.
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In eukaryotic cells many mRNAs are localized to specific regions of the cytosol, thereby allowing the local production of proteins. The process of mRNA localization can be coordinated with mRNA turnover, which can also be spatially controlled to increase the degree of mRNA localization. The coordination of mRNA localization, translation repression during transport, and mRNA degradation suggests the hypothesis that an additional layer of mRNA quality control exists in cells to degrade mRNAs that fail to be appropriately localized.
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di Penta, Alessandra, Valentina Mercaldo, Fulvio Florenzano, Sebastian Munck, M. Teresa Ciotti, Francesca Zalfa, Delio Mercanti, Marco Molinari, Claudia Bagni, and Tilmann Achsel. "Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control." Journal of Cell Biology 184, no. 3 (February 2, 2009): 423–35. http://dx.doi.org/10.1083/jcb.200807033.
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Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.
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VEDELER, Anni, and Hanne HOLLÅS. "Annexin II is associated with mRNAs which may constitute a distinct subpopulation." Biochemical Journal 348, no. 3 (June 7, 2000): 565–72. http://dx.doi.org/10.1042/bj3480565.
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Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of 32P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)+-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs.
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G�rlach, M., M. Sauter, and K. Hilse. "Specific binding of globin mRNA and distinct �-globin mRNA-segments to blotted mRNP proteins." Molecular Biology Reports 12, no. 3 (1987): 171–72. http://dx.doi.org/10.1007/bf00356883.
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Li, Yun-Ping, Janet Andersen, Arthur Zelent, Sreenivas Rao, Elisabeth Paietta, Martin S. Tallman, Peter H. Wiernik, and Robert E. Gallagher. "RARα1/RARα2-PML mRNA Expression in Acute Promyelocytic Leukemia Cells: A Molecular and Laboratory-Clinical Correlative Study." Blood 90, no. 1 (July 1, 1997): 306–12. http://dx.doi.org/10.1182/blood.v90.1.306.
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Abstract In addition to the major fusion gene PML-RARα, the t(15; 17) in acute promyelocytic leukemia (APL) produces the reciprocal fusion gene RARα-PML. To determine the scope of RARα-containing mRNA expression in APL cells, we tested PML-RARα–positive APL cells for the presence of mRNAs initiated from two distinct RARα gene promoters, α1 and α2. From the normal allele, both RARα1 and RARα2 mRNAs were expressed in all APL cases (N = 24). From the translocated allele, RARα1-PML mRNA was expressed in 77% and RARα2-PML mRNA in 28% of cases (N = 98). RARα2-PML mRNA was not observed in the absence of RARα1-PML mRNA. There was no association between RARα1-PML or RARα2-PML mRNA expression and the type of PML-RARα mRNA formed by either 5′ or 3′ breaksites in the PML gene. RARα1-PML mRNAs and RARα2-PML mRNAs from 5′ PML breaksite cases coded for full-length RARα-PML proteins but RARα2-PML mRNAs from 3′ PML breaksite cases encoded a truncated RARα2 peptide. RARα1/α2-PML mRNA expression was not associated with differences in APL cell sensitivity to all-trans retinoic acid(tRA)-induced differentiation in vitro or in clinical outcome after tRA or chemotherapy induction therapy (protocol E2491). Our analysis indicated that RARα1/α2-PML mRNA expression markedly differs from normal RARα1/α2 mRNA expression, that the difference in RARα1-PML and RARα2-PML mRNA expression frequency is primarily related to the genomic separation of the RARα1 and RARα2 coding exons, and that variations in RARα1/α2-PML mRNA expression likely have no clinically relevant function in APL cells.
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Li, Yun-Ping, Janet Andersen, Arthur Zelent, Sreenivas Rao, Elisabeth Paietta, Martin S. Tallman, Peter H. Wiernik, and Robert E. Gallagher. "RARα1/RARα2-PML mRNA Expression in Acute Promyelocytic Leukemia Cells: A Molecular and Laboratory-Clinical Correlative Study." Blood 90, no. 1 (July 1, 1997): 306–12. http://dx.doi.org/10.1182/blood.v90.1.306.306_306_312.
Full textAbstract:
In addition to the major fusion gene PML-RARα, the t(15; 17) in acute promyelocytic leukemia (APL) produces the reciprocal fusion gene RARα-PML. To determine the scope of RARα-containing mRNA expression in APL cells, we tested PML-RARα–positive APL cells for the presence of mRNAs initiated from two distinct RARα gene promoters, α1 and α2. From the normal allele, both RARα1 and RARα2 mRNAs were expressed in all APL cases (N = 24). From the translocated allele, RARα1-PML mRNA was expressed in 77% and RARα2-PML mRNA in 28% of cases (N = 98). RARα2-PML mRNA was not observed in the absence of RARα1-PML mRNA. There was no association between RARα1-PML or RARα2-PML mRNA expression and the type of PML-RARα mRNA formed by either 5′ or 3′ breaksites in the PML gene. RARα1-PML mRNAs and RARα2-PML mRNAs from 5′ PML breaksite cases coded for full-length RARα-PML proteins but RARα2-PML mRNAs from 3′ PML breaksite cases encoded a truncated RARα2 peptide. RARα1/α2-PML mRNA expression was not associated with differences in APL cell sensitivity to all-trans retinoic acid(tRA)-induced differentiation in vitro or in clinical outcome after tRA or chemotherapy induction therapy (protocol E2491). Our analysis indicated that RARα1/α2-PML mRNA expression markedly differs from normal RARα1/α2 mRNA expression, that the difference in RARα1-PML and RARα2-PML mRNA expression frequency is primarily related to the genomic separation of the RARα1 and RARα2 coding exons, and that variations in RARα1/α2-PML mRNA expression likely have no clinically relevant function in APL cells.
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Singh, Guramrit, and Zhongxia Yi. "Connections between mRNP Composition and mRNA Fate." FASEB Journal 34, S1 (April 2020): 1. http://dx.doi.org/10.1096/fasebj.2020.34.s1.00219.
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Bicknell, Alicia A., and Emiliano P. Ricci. "When mRNA translation meets decay." Biochemical Society Transactions 45, no. 2 (April 13, 2017): 339–51. http://dx.doi.org/10.1042/bst20160243.
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Messenger RNA (mRNA) translation and mRNA degradation are important determinants of protein output, and they are interconnected. Previously, it was thought that translation of an mRNA, as a rule, prevents its degradation. mRNA surveillance mechanisms, which degrade mRNAs as a consequence of their translation, were considered to be exceptions to this rule. Recently, however, it has become clear that many mRNAs are degraded co-translationally, and it has emerged that codon choice, by influencing the rate of ribosome elongation, affects the rate of mRNA decay. In this review, we discuss the links between translation and mRNA stability, with an emphasis on emerging data suggesting that codon optimality may regulate mRNA degradation.
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Kawai, Tomoko, Jinshui Fan, Krystyna Mazan-Mamczarz, and Myriam Gorospe. "Global mRNA Stabilization Preferentially Linked to Translational Repression during the Endoplasmic Reticulum Stress Response." Molecular and Cellular Biology 24, no. 15 (August 1, 2004): 6773–87. http://dx.doi.org/10.1128/mcb.24.15.6773-6787.2004.
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ABSTRACT The stability of mRNAs undergoing translation has long been a controversial question. Here, we systematically investigate links between mRNA turnover and translation during the endoplasmic reticulum (ER) stress response, a process during which protein synthesis is potently regulated. cDNA array-based approaches to assess the stability and translational status of each mRNA were devised. First, ER stress-triggered changes in mRNA stability were studied by comparing differences in steady-state mRNA levels with differences in gene transcription. Second, changes in translational status were monitored by studying ER stress-induced shifts in the relative distribution of each mRNA along sucrose gradients. Together, the array-derived data reveal complex links between mRNA stability and translation, with all regulatory groups represented: both stabilized and destabilized mRNAs were found among translationally induced as well as translationally suppressed mRNA collections. Remarkably, however, the subset of stabilized mRNAs was prominently enriched in translationally suppressed transcripts, suggesting that ER stress was capable of causing the stabilization of mRNAs associated with a global reduction in protein synthesis. The cDNA array-based approach described here can be applied to global analyses of mRNA turnover and translation and can serve to investigate subsets of mRNAs subject to joint posttranscriptional control.
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Gick, G. G., and F. Ismail-Beigi. "Thyroid hormone induction of Na(+)-K(+)-ATPase and its mRNAs in a rat liver cell line." American Journal of Physiology-Cell Physiology 258, no. 3 (March 1, 1990): C544—C551. http://dx.doi.org/10.1152/ajpcell.1990.258.3.c544.
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The expression of mRNAs encoding the alpha- and beta-subunits of Na(+)-K(+)-ATPase (Na(+)-K+ pump) was examined in a rat liver cell line, Clone 9, in various thyroidal states. Northern blot analysis of total RNA isolated from cells incubated in hypothyroid serum-containing medium revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1-(mRNA alpha 1) and beta- (mRNA beta) subunits; mRNAs encoding the alpha 2- and alpha 3-subunits were undetectable. There was a discrepancy in the abundance of mRNA alpha 1 relative to mRNA beta such that mRNA alpha 1 exceeded the sum of the multiple mRNA beta bands by approximately 35-fold. 3,3',5-Triiodothyronine (T3) produced a coordinate augmentation of mRNA alpha 1 and mRNA beta contents that was demonstrable within 2 h and preceded the stimulation of Na(+)-K(+)-ATPase activity. After incubation of cells with T3 for 48 h, Na(+)-K(+)-ATPase activity was stimulated by 1.32-fold, whereas mRNA alpha 1 and mRNA beta abundances were increased 1.46- and 2.87-fold, respectively. Treatment of cells for 6 h with 10 micrograms/ml cycloheximide, a concentration sufficient to inhibit protein synthesis by 95%, elicited a 3.5- and 5.1-fold increase in mRNA alpha 1 and mRNA beta content, respectively. Cycloheximide abrogated the stimulatory effect of T3 on mRNA beta abundance, whereas the T3-induced increase in mRNA alpha 1 content was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)
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Aisina, Dana, Raigul Niyazova, Shara Atambayeva, and Anatoliy Ivashchenko. "Prediction of clusters of miRNA binding sites in mRNA candidate genes of breast cancer subtypes." PeerJ 7 (November 13, 2019): e8049. http://dx.doi.org/10.7717/peerj.8049.
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The development of breast cancer (BC) subtypes is controlled by distinct sets of candidate genes, and the expression of these genes is regulated by the binding of their mRNAs with miRNAs. Predicting miRNA associations and target genes is thus essential when studying breast cancer. The MirTarget program identifies the initiation of miRNA binding to mRNA, the localization of miRNA binding sites in mRNA regions, and the free energy from the binding of all miRNA nucleotides with mRNA. Candidate gene mRNAs have clusters (miRNA binding sites with overlapping nucleotide sequences). mRNAs of EPOR, MAZ and NISCH candidate genes of the HER2 subtype have clusters, and there are four clusters in mRNAs of MAZ, BRCA2 and CDK6 genes. Candidate genes of the triple-negative subtype are targets for multiple miRNAs. There are 11 sites in CBL mRNA, five sites in MMP2 mRNA, and RAB5A mRNA contains two clusters in each of the three sites. In SFN mRNA, there are two clusters in three sites, and one cluster in 21 sites. Candidate genes of luminal A and B subtypes are targets for miRNAs: there are 21 sites in FOXA1 mRNA and 15 sites in HMGA2 mRNA. There are clusters of five sites in mRNAs of ITGB1 and SOX4 genes. Clusters of eight sites and 10 sites are identified in mRNAs of SMAD3 and TGFB1 genes, respectively. Organizing miRNA binding sites into clusters reduces the proportion of nucleotide binding sites in mRNAs. This overlapping of miRNA binding sites creates a competition among miRNAs for a binding site. From 6,272 miRNAs studied, only 29 miRNAs from miRBase and 88 novel miRNAs had binding sites in clusters of target gene mRNA in breast cancer. We propose using associations of miRNAs and their target genes as markers in breast cancer subtype diagnosis.
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Holmquist, Chris E., Wenxia He, Rita M. Meganck, and William F. Marzluff. "Knockouts of TUT7 and 3′hExo show that they cooperate in histone mRNA maintenance and degradation." RNA 28, no. 11 (August 30, 2022): 1519–33. http://dx.doi.org/10.1261/rna.079233.122.
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Metazoan histone mRNAs are the only cellular eukaryotic mRNAs that are not polyadenylated, ending instead in a conserved stem–loop. SLBP is bound to the 3′ end of histone mRNAs and is required for translation of histone mRNA. The expression of histone mRNAs is tightly cell-cycle regulated. A major regulatory step is rapid degradation of histone mRNA at the end of S-phase or when DNA synthesis is inhibited in S-phase. 3′hExo, a 3′ to 5′ exonuclease, binds to the SLBP/SL complex and trims histone mRNA to 3 nt after the stem–loop. Together with a terminal uridyl transferase, 3′hExo maintains the length of the histone mRNA during S-phase. 3′hExo is essential for initiating histone mRNA degradation on polyribosomes, initiating degradation into the 3′ side of the stem–loop. There is extensive uridylation of degradation intermediates in the 3′ side of the stem when histone mRNA is degraded. Here, we knocked out TUT7 and 3′hExo and we show that both modification of histone mRNA during S-phase and degradation of histone mRNA involve the interaction of 3′hExo, and a specific TUTase, TENT3B (TUT7, ZCCHC6). Knockout of 3′hExo prevents the initiation of 3′ to 5′ degradation, stabilizing histone mRNA, whereas knockout of TUT7 prevents uridylation of the mRNA degradation intermediates, slowing the rate of degradation. In synchronized 3′hExo KO cells, histone mRNA degradation is delayed, but the histone mRNA is degraded prior to mitosis by a different pathway.
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Yang, Edward, Erik van Nimwegen, Mihaela Zavolan, Nikolaus Rajewsky, Mark Schroeder, Marcelo Magnasco, and James E. Darnell. "Decay Rates of Human mRNAs: Correlation With Functional Characteristics and Sequence Attributes." Genome Research 13, no. 8 (August 2003): 1863–72. http://dx.doi.org/10.1101/gr.1272403.
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Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions. We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species. Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes. The results show statistically significant organizational principles in the variation of decay rates among functional classes. In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in “fast-decaying” mRNAs with half-lives <2 h. In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs. Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes. Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript. Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay. However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites.
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Fernandes, Nikita, and J. Ross Buchan. "RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly." Nucleic Acids Research 48, no. 11 (May 12, 2020): 6265–79. http://dx.doi.org/10.1093/nar/gkaa352.
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Abstract P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3′UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3′UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3′UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA ‘scaffolds’. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3′UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.
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Gong, Chenguang, Yalan Tang, and Lynne E. Maquat. "mRNA–mRNA duplexes that autoelicit Staufen1-mediated mRNA decay." Nature Structural & Molecular Biology 20, no. 10 (September 22, 2013): 1214–20. http://dx.doi.org/10.1038/nsmb.2664.
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Hall, C., C. M. Lowndes, T. K. Leung, D. N. Cooper, A. M. Goate, and L. Lim. "Expression and developmental regulation of two unique mRNAs specific to brain membrane-bound polyribosomes." Biochemical Journal 244, no. 2 (June 1, 1987): 359–66. http://dx.doi.org/10.1042/bj2440359.
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Translation in vitro of membrane-bound polyribosomal mRNAs from rat brain has shown several to be developmentally regulated [Hall & Lim (1981) Biochem. J. 196, 327-336]. Here we describe the isolation and characterization of cDNAs corresponding to two such brain mRNAs. One cDNA (M444) hybrid-selected a 0.95 kb mRNA directing the synthesis in vitro of a 21 kDa pI-6.3 polypeptide, which was processed in vitro by microsomal membranes. A second cDNA (M1622) hybridized to a 2.2 kb mRNA directing the synthesis of a 55 kDa pI-5.8 polypeptide. Both mRNAs were specific to membrane-bound polyribosomes. Restriction maps of the corresponding genomic DNA sequences are consistent with both being single copy. The two mRNAs were present in astrocytic and neuronal cultures, but not in liver or spleen or in neuroblastoma or glioma cells. The two mRNAs were differently regulated during brain development. In the developing forebrain there was a gradual and sustained increase in M444 mRNA during the first 3 weeks post partum, whereas M1622 mRNA appeared earlier and showed no further increase after day 10. In the cerebellum the developmental increase in M444 mRNA was biphasic. After a small initial increase there was a decrease in this mRNA at day 10, coincident with high amounts of M1622 mRNA. This was followed by a second, larger, increase in M444 mRNA, when amounts of M1622 mRNA were constant. The contrasting changes in these two mRNAs in the developing cerebellum are of particular interest, since they occur during an intensive period of cell proliferation, migration and altering neural connectivity. As these mRNAs are specific to differentiated neural tissue, they represent useful molecular markers for studying brain differentiation.
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D'Agostino, D. M., B. K. Felber, J. E. Harrison, and G. N. Pavlakis. "The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs." Molecular and Cellular Biology 12, no. 3 (March 1992): 1375–86. http://dx.doi.org/10.1128/mcb.12.3.1375-1386.1992.
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Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.
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D'Agostino, D. M., B. K. Felber, J. E. Harrison, and G. N. Pavlakis. "The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs." Molecular and Cellular Biology 12, no. 3 (March 1992): 1375–86. http://dx.doi.org/10.1128/mcb.12.3.1375.
Full textAbstract:
Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.
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Wang, Ke, Changping Yin, Xian Du, Suli Chen, Jianshu Wang, Li Zhang, Lantian Wang, et al. "A U2-snRNP–independent role of SF3b in promoting mRNA export." Proceedings of the National Academy of Sciences 116, no. 16 (March 28, 2019): 7837–46. http://dx.doi.org/10.1073/pnas.1818835116.
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To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3′ processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b–THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP–independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.
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Shapiro, R. A., D. Herrick, R. E. Manrow, D. Blinder, and A. Jacobson. "Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates." Molecular and Cellular Biology 8, no. 5 (May 1988): 1957–69. http://dx.doi.org/10.1128/mcb.8.5.1957-1969.1988.
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As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.
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Shapiro, R. A., D. Herrick, R. E. Manrow, D. Blinder, and A. Jacobson. "Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates." Molecular and Cellular Biology 8, no. 5 (May 1988): 1957–69. http://dx.doi.org/10.1128/mcb.8.5.1957.
Full textAbstract:
As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.
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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.
Full textAbstract:
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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Segal, Scott P., Travis Dunckley, and Roy Parker. "Sbp1p Affects Translational Repression and Decapping in Saccharomyces cerevisiae." Molecular and Cellular Biology 26, no. 13 (July 1, 2006): 5120–30. http://dx.doi.org/10.1128/mcb.01913-05.
Full textAbstract:
ABSTRACT The relationship between translation and mRNA turnover is critical to the regulation of gene expression. One major pathway for mRNA turnover occurs by deadenylation, which leads to decapping and subsequent 5′-to-3′ degradation of the body of the mRNA. Prior to mRNA decapping, a transcript exits translation and enters P bodies to become a potential decapping substrate. To understand the transition from translation to decapping, it is important to identify the factors involved in this process. In this work, we identify Sbp1p (formerly known as Ssb1p), an abundant RNA binding protein, as a high-copy-number suppressor of a conditional allele in the decapping enzyme. Sbp1p overexpression restores normal decay rates in decapping-defective strains and increases P-body size and number. In addition, Sbp1p promotes translational repression of mRNA during glucose deprivation. Moreover, P-body formation is reduced in strains lacking Sbp1p. Sbp1p acts in conjunction with Dhh1p, as it is required for translational repression and P-body formation in pat1Δ strains under these conditions. These results identify Sbp1p as a new protein that functions in the transition of mRNAs from translation to an mRNP complex destined for decapping.
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Sun, D. Y., J. Z. Guo, H. A. Hartmann, H. Uno, and L. E. Hokin. "Differential expression of the alpha 2 and beta messenger RNAs of Na,K-ATPase in developing brine shrimp as measured by in situ hybridization." Journal of Histochemistry & Cytochemistry 40, no. 4 (April 1992): 555–62. http://dx.doi.org/10.1177/40.4.1313064.
Full textAbstract:
We used in situ hybridization histochemistry with synthetic oligonucleotide probes to localize the mRNAs encoding the alpha 2- and beta-mRNAs of Na,K-ATPase during development of the brine shrimp Artemia. The mRNAs of the alpha 2- and beta-subunit were of low abundance in the cysts; in addition, less mRNA of the beta-subunit was localized. During emergence (12 hr), there was an increase in alpha 2-subunit mRNA in the gut mucosa, but there was a burst in beta-subunit mRNA throughout. As development progressed, the mRNAs of both the alpha 2- and beta-subunits showed a distinct pattern of expression in which the mRNA in the salt gland was of greatest abundance, followed by epidermal cells and gut mucosa. After 36 hr the alpha 2-subunit mRNA began to decrease in all positive cells but still remained highest in the salt gland and the brain region, while the mRNA of the beta-subunit kept increasing in the gut mucosa. Finally, the greatest abundance of the beta-subunit mRNA shifted from the salt gland to the antenna gland and the epidermal cells in the tail region, but the alpha 2-subunit mRNA did not. The more widespread distribution of the beta-mRNA than alpha 2-mRNA at certain stages (e.g., there was no alpha 2-mRNA in the antenna gland at the adult stage) is in all likelihood due to the marked drop in the alpha 2-subunit and a rise in alpha 1-subunit previously seen by Peterson et al. on polyacrylamide gel electrophoresis, as development progresses.
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van Zalen, Sebastiaan, and J. Eric Russell. "Two Novel Trans-Acting Factors Dictate the High Cytoplasmic Stability of β-Globin mRNA." Blood 116, no. 21 (November 19, 2010): 644. http://dx.doi.org/10.1182/blood.v116.21.644.644.
Full textAbstract:
Abstract Abstract 644 The efficient accumulation of hemoglobin in mature erythrocytes is critically dependent upon the high stabilities of mRNAs encoding human α- and β-globin proteins. These mRNAs are likely to be stabilized by interactions between one or more trans-acting regulatory factors that target defined cis-acting elements within their 3′UTRs. Several ubiquitous factors that are known to bind to the β-globin 3′UTR (including αCP, PTBP1, and nucleolin) are largely restricted to the nucleus and therefore unlikely to contribute to regulatory processes affecting β-globin mRNA in the cytoplasm. Consequently, we conducted a series of experiments that identify and characterize mRNA-binding factors that dictate the properties of β-globin mRNA in the cytoplasm of erythroid progenitor cells. Using electrophoretic gel mobility shift analyses (EMSA), we defined a characteristic mRNP complex that assembles on the β-globin 3′UTR in cytoplasmic extract–but not nuclear extract–prepared from erythroid K562 cells. This mRNP ‘β-complex’ appears to be erythroid-specific, as it fails to assemble in extracts prepared from non-erythroid HeLa or HEK cells. The 3′UTR binding site for the β-complex was identified using an EMSA-competition approach; remarkably, the target sequence is encompassed within a 12-nt region previously identified as a functional determinant of β-globin mRNA stability in in vivo analyses. Additional experiments fine-mapped the β-complex binding site to a GGGGG pentanucleotide motif within the mRNA-stabilizing region. The functional importance of the pentanucleotide was illustrated by mRNA decay experiments in intact erythroid K562 cells showing that full-length β-globin mRNAs are destabilized by introduction of the same GGGGG->CCGGG mutation that ablates β-complex assembly in EMSA analyses. To identify trans-factors that comprise the β-complex, we performed affinity chromatography using ssDNA probes corresponding to the β-complex binding motif. The native 3′UTR probe retained 42- and 47-kDa proteins, while a probe carrying the CCGGG mutation failed to bind either factor. Subsequent LC/MS/MS analyses identified the two proteins as YB-1 and AUF-1. The identities of these two mRNA-binding factors, which have previously been implicated in the post-transcriptional regulation of heterologous mRNAs, were subsequently confirmed by immunoblot of the protein-DNA complexes. Subsequent analyses suggested a functional role for both factors: EMSA supershift experiments confirmed that YB-1 is a component of the β-complex, and RNA immunoprecipitation analyses demonstrated that both YB-1 and AUF-1 specifically bind to β-globin mRNA in vivo in intact erythroid K562 cells. Collectively, these data identify two novel trans-acting factors that bind to cytoplasmic β-globin mRNA in an erythroid-specific fashion, at a site that dictates its stability in intact cells. We are currently engaged in siRNA knock-down experiments to validate experiments that suggest the importance of these trans-acting factors to the constitutive cytoplasmic stability of β-globin mRNA, as well as structural analyses intended to define RNA-protein and protein-protein interactions that are critical to normal functioning of the β-complex. The results of these experiments have obvious implications for the design of novel therapies for patients with congenital disorders of β-globin gene expression, including sickle cell disease and β thalassemia. Disclosures: No relevant conflicts of interest to declare.
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Naora, H., and IG Young. "Mechanisms regulating the mRNA levels of interleukin-5 and two other coordinately expressed lymphokines in the murine T lymphoma EL4.23." Blood 83, no. 12 (June 15, 1994): 3620–28. http://dx.doi.org/10.1182/blood.v83.12.3620.3620.
Full textAbstract:
Abstract The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13- acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear run-off analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.
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Naora, H., and IG Young. "Mechanisms regulating the mRNA levels of interleukin-5 and two other coordinately expressed lymphokines in the murine T lymphoma EL4.23." Blood 83, no. 12 (June 15, 1994): 3620–28. http://dx.doi.org/10.1182/blood.v83.12.3620.bloodjournal83123620.
Full textAbstract:
The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor (GM- CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13- acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear run-off analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.