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Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor

ABSTRACT: Translational readthrough generates proteins with extended C terminus which often possess distinct properties. Here, we have demonstrated translational readthrough of AGO1 using various reporter assays. Analysis of ribosome profiling data and mass-spectrometry data provided additional evidences for translational readthrough of AGO1. We could also detect endogenous readthrough product, Ago1x, using a specific antibody both in vitro and in vivo. This process is programmed by a cis sequence downstream to the canonical stop codon of AGO1 which could drive readthrough even in a heterologous context. The cis sequence has a let-7a miRNA binding site and the readthrough process is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.

SUBMITTER: Anumeha Singh

PROVIDER: S-SCDT-EMBOJ-2018-100727 | biostudies-other |

REPOSITORIES: biostudies-other

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Let-7a-regulated translational readthrough of mammalian AGO1 generates a microRNA pathway inhibitor.

Project description:Translational readthrough generates proteins with extended C-termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let-7a miRNA-binding site, and readthrough is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.

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Programmed translational readthrough generates antiangiogenic VEGF-Ax.

Project description:Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

| S-EPMC4113015 | biostudies-literature

Cellular senescence limits translational readthrough.

Project description:The origin and evolution of cancer cells is considered to be mainly fueled by DNA mutations. Although translation errors could also expand the cellular proteome, their role in cancer biology remains poorly understood. Tumor suppressors called caretakers block cancer initiation and progression by preventing DNA mutations and/or stimulating DNA repair. If translational errors contribute to tumorigenesis, then caretaker genes should prevent such errors in normal cells in response to oncogenic stimuli. Here, we show that the process of cellular senescence induced by oncogenes, tumor suppressors or chemotherapeutic drugs is associated with a reduction in translational readthrough (TR) measured using reporters containing termination codons withing the context of both normal translation termination or programmed TR. Senescence reduced both basal TR and TR stimulated by aminoglycosides. Mechanistically, the reduction of TR during senescence is controlled by the RB tumor suppressor pathway. Cells that escape from cellular senescence either induced by oncogenes or chemotherapy have an increased TR. Also, breast cancer cells that escape from therapy-induced senescence express high levels of AGO1x, a TR isoform of AGO1 linked to breast cancer progression. We propose that senescence and the RB pathway reduce TR limiting proteome diversity and the expression of TR proteins required for cancer cell proliferation.

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The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila.

Project description:The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into approximately 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. In this study, we characterize Drosophila small RNAs that derive from short intronic hairpins termed "mirtrons." Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and we provide evidence that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs.

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Correction: Functional Translational Readthrough: A Systems Biology Perspective.

Project description:[This corrects the article DOI: 10.1371/journal.pgen.1006196.].

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Novel functional small RNAs are selectively loaded onto mammalian Ago1.

Project description:Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1-4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein-Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1-4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

| S-EPMC4005649 | biostudies-literature

microRNA-mediated messenger RNA deadenylation contributes to translational repression in mammalian cells.

Project description:Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3' untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3' poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3' UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs.

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Yeast applied readthrough inducing system (YARIS): an invivo assay for the comprehensive study of translational readthrough.

Project description:Stop codon readthrough-the decoding of a stop codon by a near-cognate tRNA-is employed by viruses to balance levels of enzymatic and structural proteins and by eukaryotic cells to enable isoform-specific protein synthesis in response to external stimuli. Owing to the prevalence of premature termination codons in human disease, readthrough has emerged as an attractive therapeutic target. A growing list of various features, for example the +4 nucleotide immediately following the stop codon, modulate readthrough levels, underscoring the need for systematic investigation of readthrough. Here, we identified and described a complete group of yeast tRNAs that induce readthrough in the stop-codon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs). These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporter-based assay enabling simultaneous detection of readthrough levels at all twelve stop-codon tetranucleotides and as a function of the complete set of rti-tRNAs. We demonstrate the utility of YARIS for systematic study of translation readthrough by employing it to interrogate the effects of natural rti-tRNA modifications, as well as various readthrough-inducing drugs (RTIDs). This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.

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Dbp5/DDX19 between Translational Readthrough and Nonsense Mediated Decay.

Project description:The DEAD-box protein Dbp5 (human DDX19) remodels RNA-protein complexes. Dbp5 functions in ribonucleoprotein export and translation termination. Termination occurs, when the ribosome has reached a stop codon through the Dbp5 mediated delivery of the eukaryotic termination factor eRF1. eRF1 contacts eRF3 upon dissociation of Dbp5, resulting in polypeptide chain release and subsequent ribosomal subunit splitting. Mutations in DBP5 lead to stop codon readthrough, because the eRF1 and eRF3 interaction is not controlled and occurs prematurely. This identifies Dbp5/DDX19 as a possible potent drug target for nonsense suppression therapy. Neurodegenerative diseases and cancer are caused in many cases by the loss of a gene product, because its mRNA contained a premature termination codon (PTC) and is thus eliminated through the nonsense mediated decay (NMD) pathway, which is described in the second half of this review. We discuss translation termination and NMD in the light of Dbp5/DDX19 and subsequently speculate on reducing Dbp5/DDX19 activity to allow readthrough of the PTC and production of a full-length protein to detract the RNA from NMD as a possible treatment for diseases.

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Robustness by intrinsically disordered C-termini and translational readthrough.

Project description:During protein synthesis genetic instructions are passed from DNA via mRNA to the ribosome to assemble a protein chain. Occasionally, stop codons in the mRNA are bypassed and translation continues into the untranslated region (3'-UTR). This process, called translational readthrough (TR), yields a protein chain that becomes longer than would be predicted from the DNA sequence alone. Protein sequences vary in propensity for translational errors, which may yield evolutionary constraints by limiting evolutionary paths. Here we investigated TR in Saccharomyces cerevisiae by analysing ribosome profiling data. We clustered proteins as either prone or non-prone to TR, and conducted comparative analyses. We find that a relatively high frequency (5%) of genes undergo TR, including ribosomal subunit proteins. Our main finding is that proteins undergoing TR are highly expressed and have intrinsically disordered C-termini. We suggest that highly expressed proteins may compensate for the deleterious effects of TR by having intrinsically disordered C-termini, which may provide conformational flexibility but without distorting native function. Moreover, we discuss whether minimizing deleterious effects of TR is also enabling exploration of the phenotypic landscape of protein isoforms.

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