Challenging the Roles of NSP3 and Untranslated Regions in Rotavirus mRNA Translation.
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ABSTRACT:
SUBMITTER: Gratia M
PROVIDER: S-EPMC4699793 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We present eIF3a PAR-iCLIP data which demonstrate that eIF3 predominantly binds mRNAs within the 5'UTR. eIF3 binding sites are also in proximity to m6A residues within the 5'UTR of cellular mRNAs. Two replicates of eIF3a PAR-iCLIP in HEK293T cells.
2015-10-26 | E-GEOD-73405 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We performed m6A profiling in cells subjected to heat shock stress and observed increased 5'UTR methylation after heat shock. For these studies, we used single-nucleotide resolution m6A mapping (miCLIP), which enables the detection of m6A residues separately from m6Am residues. Thus, the goal of these experiments was to identify m6A residues which are increased in the 5'UTR after heat shock stress.
2015-10-26 | GSE73405 | GEO
Project description:Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3'-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.
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Project description:Combining a genetic and pharmacological approach, we modulated eIF4E activity across its physiological activity range and identified subsets of genes whose translation was either hypo- or hyper- sensitive to eIF4E activity changes. eIF4E hypersensitive genes had longer 5'UTRs with higher GC content; and longer 3'UTRs with lower GC content and a higher density of unique microRNA target sites. Proliferation related genes were enriched among eIF4E hypersensitive genes; and consistent with this, decreasing eIF4E activity inhibited cell cycle transit. Our findings provide genome wide insights into the properties of mRNAs under translational control across the physiological range of eIF4E activity. NIH 3T3 derivatives genetically altered to induce eIF4E when treated with mifepristone and a non-inducible control NIH 3T3 derivative were cultured with or without treatment with mifepristone and varying doses of a pharmacological eIF4E inhibiting agent, 4Ei-1 (0 μM, 10 μM, 50 μM, 100 μM, 200 μM). Total RNA and polyribosome RNA were isolated after 4h of treatment. Three replicates of each experimental group were completed. In addition, three replicates of the inducible cell line treated with less potent analog to the inhibiting agent, 4Ei-4, were completed to probe for non-specific drug effects.
2015-12-31 | E-GEOD-56072 | biostudies-arrayexpress
Project description:Combining a genetic and pharmacological approach, we modulated eIF4E activity across its physiological activity range and identified subsets of genes whose translation was either hypo- or hyper- sensitive to eIF4E activity changes. eIF4E hypersensitive genes had longer 5'UTRs with higher GC content; and longer 3'UTRs with lower GC content and a higher density of unique microRNA target sites. Proliferation related genes were enriched among eIF4E hypersensitive genes; and consistent with this, decreasing eIF4E activity inhibited cell cycle transit. Our findings provide genome wide insights into the properties of mRNAs under translational control across the physiological range of eIF4E activity.
2015-12-31 | GSE56072 | GEO
Project description:The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3' UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5' or 3' UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3' UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3' UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3' UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3' UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3' UTR, but not the edited or deletion mutant NP 3' UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3' UTR, VP40 3' UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3' UTR edits also enhanced translation, but neither the wild-type nor the edited 3' UTRs induced IFN. These findings implicate filoviral mRNA 3' UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3' UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3' UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3' UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.
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Project description:BACKGROUND: Deliberate cellular reprogramming is becoming a realistic objective in the clinic. While the origin of the target cells is critical, delivery of bioactive molecules to trigger a shift in cell-fate remains the major hurdle. To date, several strategies based either on non-integrative vectors, protein transfer or mRNA delivery have been investigated. In a recent study, a unique modification in the retroviral genome was shown to enable RNA transfer and its expression. RESULTS: Here, we used the retroviral mRNA delivery approach to study the impact of modifying gene-flanking sequences on RNA transfer. We designed modified mRNAs for retroviral packaging and used the quantitative luciferase assay to compare mRNA expression following viral transduction of cells. Cloning the untranslated regions of the vimentin or non-muscular myosin heavy chain within transcripts improved expression and stability of the reporter gene while slightly modifying reporter-RNA retroviral delivery. We also observed that while the modified retroviral platform was the most effective for retroviral mRNA packaging, the highest expression in target cells was achieved by the addition of a non-viral UTR to mRNAs containing the packaging signal. CONCLUSIONS: Through molecular engineering we have assayed a series of constructs to improve retroviral mRNA transfer. We showed that an authentic RNA retroviral genomic platform was most efficiently transferred but that adding UTR sequences from highly expressed genes could improve expression upon transfection while having only a slight effect on expression from transferred RNA. Together, these data should contribute to the optimisation of retroviral mRNA-delivery systems that test combinations of UTRs and packaging platforms.
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Project description:The serotonin transporter (SERT, SLC6A4) is a Na+-dependent transporter that regulates the availability of serotonin (5-HT, 5-hydroxytryptamine), a key neurotransmitter and hormone in the brain and the intestine. The human SERT gene consists of two alternate promoters that drive expression of an identical SERT protein. However, there are different mRNA transcript variants derived from these two promoters that differ in their 5' untranslated region (5'UTR), which is the region of the mRNA upstream from the protein-coding region. Two of these transcripts contain exon-1a and are abundant in neuronal tissue, whereas the third transcript contains exon-1c and is abundant in the intestine. The 3'UTR is nearly identical among the transcripts. Current studies tested the hypothesis that the UTRs of SERT influence its expression in intestinal epithelial cells (IECs) by controlling mRNA or protein levels. The SERT UTRs were cloned into luciferase reporter plasmids and luciferase mRNA and activity were measured following transient transfection of the UTR constructs into the model IEC Caco-2. Luciferase activity and mRNA abundance were higher than the empty vector for two of the three 5'UTR variants. Calculation of translation index (luciferase activity divided by the relative luciferase mRNA level) revealed that the exon-1a containing 5'UTRs had enhanced translation when compared to the exon-1c containing 5'UTR which exhibited a low translation efficiency. Compared to the empty vector, the SERT 3'UTR markedly decreased luciferase activity. In silico analysis of the SERT 3'UTR revealed many conserved potential miRNA binding sites that may be responsible for this decrease. In conclusion, we have shown that the UTRs of SERT regulate mRNA abundance and protein expression. Delineating the molecular basis by which the UTRs of SERT influence its expression will lead to an increased understanding of post-transcriptional regulation of SERT in GI disorders associated with altered 5-HT availability.
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