Cotranscriptional effect of a premature termination codon revealed by live-cell imaging

Turris, Valeria de; Nicholson, Pamela; Orozco, Rodolfo Zamudio; Singer, Robert H.; Mühlemann, Oliver

doi:10.1261/rna.02918111

Cited by 44 publications

(42 citation statements)

References 64 publications

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“…Given the regulatory role of uORFs in some coding transcripts (Arribere and Gilbert, 2013;Calvo et al, 2009;Hinnebusch, 2005;Hood et al, 2009;Johansson and Jacobson, 2010), 5Ј leader-like translation might affect lncRNA stability and/or subcellular localization. Translating ORFs within lncRNAs might target the transcript for nonsensemediated decay (Tani et al, 2013), degrading it in the cytoplasm and/or retaining it in the nucleus (de Turris et al, 2011), resulting in the predominantly nuclear localization of most lncRNAs (Derrien et al, 2012). Prime candidates for such regulation are the minORF-…”

Section: Toc Refines Classification Of Lncrnasmentioning

confidence: 99%

Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs

et al. 2013

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SUMMARYLarge-scale genomics and computational approaches have identified thousands of putative long non-coding RNAs (lncRNAs). It has been controversial, however, as to what fraction of these RNAs is truly non-coding. Here, we combine ribosome profiling with a machine-learning approach to validate lncRNAs during zebrafish development in a high throughput manner. We find that dozens of proposed lncRNAs are protein-coding contaminants and that many lncRNAs have ribosome profiles that resemble the 5Ј leaders of coding RNAs. Analysis of ribosome profiling data from embryonic stem cells reveals similar properties for mammalian lncRNAs. These results clarify the annotation of developmental lncRNAs and suggest a potential role for translation in lncRNA regulation. In addition, our computational pipeline and ribosome profiling data provide a powerful resource for the identification of translated open reading frames during zebrafish development.

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Section: Toc Refines Classification Of Lncrnasmentioning

confidence: 99%

Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs

et al. 2013

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“…Proteins were transferred to Optitran BA-S 85 reinforced nitrocellulose membranes (Whatman GmbH) and probed with the rabbit polyclonal antibodies anti-hUPF1 (1:1000, RENT1 H-300, sc-48802, Santa Cruz Biotechnology or Lykke- Andersen et al 2000), anti-hUPF2, anti-hUPF3b (both: 1:3000) (Lykke- Andersen et al 2000), anti-hSMG5 (1:2000) (Franks et al 2010), anti-hSMG6 (1:150, custom-made for our laboratory by Eurogentec; see de Turris et al 2011), anti-hSMG7 (for Figs. 4C and 5A: 1:500 dilution of crude serum from a rabbit immunized with a bacterially expressed His-tagged polypeptide comprising the C-terminal amino acids 870-1137 of hSMG7; for Fig.…”

Section: Western Blottingmentioning

confidence: 99%

Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

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Ruepp

et al. 2013

RNA

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Nonsense-mediated mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation mechanism that eliminates mRNAs with the termination codon (TC) located in an unfavorable environment for efficient translation termination. The best-studied NMD-targeted mRNAs contain premature termination codons (PTCs); however, NMD regulates even many physiological mRNAs. An exon-junction complex (EJC) located downstream from a TC acts as an NMD-enhancing signal, but is not generally required for NMD. Here, we compared these "EJC-enhanced" and "EJC-independent" modes of NMD with regard to their requirement for seven known NMD factors in human cells using two well-characterized NMD reporter genes (immunoglobulin μ and β-Globin) with or without an intron downstream from the PTC. We show that both NMD modes depend on UPF1 and SMG1, but detected transcript-specific differences with respect to the requirement for UPF2 and UPF3b, consistent with previously reported UPF2-and UPF3-independent branches of NMD. In addition and contrary to expectation, a higher sensitivity of EJC-independent NMD to reduced UPF2 and UPF3b concentrations was observed. Our data further revealed a redundancy of the endo-and exonucleolytic mRNA degradation pathways in both modes of NMD. Moreover, the relative contributions of both decay pathways differed between the reporters, with PTC-containing immunoglobulin μ transcripts being preferentially subjected to SMG6-mediated endonucleolytic cleavage, whereas β-Globin transcripts were predominantly degraded by the SMG5/SMG7-dependent pathway. Overall, the surprising heterogeneity observed with only two NMD reporter pairs suggests the existence of several mechanistically distinct branches of NMD in human cells.

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“…In support of this possibility, it was reported that the inhibition of nuclear export does not decrease the decay of nucleusassociated PTC-containing mRNAs (Bü hler et al 2002); that some ribosome components in yeast accumulate at the site of PTC-containing gene transcription (Brogna et al 2002); that in mammalian cells, translation can occur in the nucleoplasm and in the nucleolus (David et al 2012); and that PTC-containing immunoglobulin (Ig)-m and T-cell receptor-b transcripts, but not their PTC-free counterparts, are retained at their transcription sites (Mü hlemann et al 2001;de Turris et al 2011). Thus, whether nucleus-associated NMD is a consequence of nuclear or cytoplasmic translation followed by rapid degradation is still questioned (Dahlberg and Lund 2004;Reid and Nicchitta 2012).…”

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confidence: 99%

Temporal and spatial characterization of nonsense-mediated mRNA decay

et al. 2013

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Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for ''surveying'' mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTCcontaining b-globin mRNA in intact single cells after the induction of b-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing b-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free b-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.

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Cotranscriptional effect of a premature termination codon revealed by live-cell imaging

Cited by 44 publications

References 64 publications

Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs

Ribosome profiling reveals resemblance between long non-coding RNAs and 5′ leaders of coding RNAs

Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

Temporal and spatial characterization of nonsense-mediated mRNA decay

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