A comprehensive coverage insurance for cells: revealing links between ribosome collisions, stress responses and mRNA surveillance

Cells of metazoans respond to internal and external stressors by activating stress response pathways that aim for re-establishing cellular homoeostasis or, if this cannot be achieved, triggering programmed cell death. Problems during translation, arising from defective mRNAs, tRNAs, ribosomes or protein misfolding, can activate stress response pathways as well as mRNA surveillance and ribosome quality control programs. Recently, ribosome collisions have emerged as a central signal for translational stress and shown to elicit different stress responses. Here, we review our current knowledge about the intricate mutual connections between ribosome collisions, stress response pathways and mRNA surveillance. A central factor connecting the sensing of collided ribosomes with degradation of the nascent polypeptides, dissociation of the stalled ribosomes and degradation of the mRNA by no-go or non-stop decay is the E3-ligase ZNF598. We tested whether ZNF598 also plays a role in nonsense-mediated mRNA decay (NMD) but found that it is dispensable for this translation termination-associated mRNA surveillance pathway, which in combination with other recent data argues against stable ribosome stalling at termination codons being the NMD-triggering signal

Efficient downregulation of immunoglobulin μ mRNA with premature translation-termination codons requires the 5′-half of the VDJ exon

Premature translation-termination codons (PTCs) elicit rapid degradation of the mRNA by a process called nonsense-mediated mRNA decay (NMD). NMD appears to be significantly more efficient for mRNAs of genes belonging to the immunoglobulin superfamily, which frequently acquire PTCs during VDJ rearrangment, than for mRNAs of other genes. To identify determinants for efficient NMD, we developed a minigene system derived from a mouse immunoglobulin μ gene (Ig-μ) and measured the effect of PTCs at different positions on the mRNA level. This revealed that PTCs located downstream of the V-D junction in the VDJ exon of Ig-μ minigenes and of endogenous Ig-μ genes elicit very strong mRNA downregulation, whereas NMD efficiency decreases gradually further upstream in the V segment where a PTC was inserted. Interestingly, two PTCs are in positions where they usually do not trigger NMD (<50 nt from the 3′-most 5′ splice site) still resulted in reduced mRNA levels. Using a set of hybrid constructs comprised of Ig-μ and an inefficient substrate for NMD, we identified a 177 nt long element in the V segment that is necessary for efficient downregulation of PTC-containing hybrid transcripts. Moreover, deletion of this NMD-promoting element from the Ig-μ minigene results in loss of strong NM

What makes an NMD target? Identification and characterization of NMD sensitive endogenous transcripts in human cells

Nonsense-mediated mRNA decay (NMD) was initially described as a quality control mechanism clearing transcripts harboring a premature termination codon (PTC) from the cell. Because a PTC can be caused by a mutation in the gene sequence or by aberrant pre-mRNA splicing, NMD was always associated with abnormal or pathological conditions. However, transcriptome profiling revealed that many mRNAs with no PTC are downregulated, often only moderately, by the NMD pathway. NMD therefore emerges as a post-transcriptional mechanism contributing to the fine-tuning of gene expression. The molecular mechanism of NMD is only partially understood and depends on the concerted activity of many factors, but accumulating evidence indicates that aberrant translation termination is the general underlying condition triggering NMD. Previous genome-wide studies showed little agreement regarding the endogenous transcripts subject to NMD, and depletion of different NMD factors affected different sets of transcripts. To address these inconsistencies, we performed RNA-seq experiments following the latest best practices of the field. We carried out knockdowns of three well-characterized NMD factors (UPF1, SMG6 and SMG7) and also operated the respective rescues, which allowed us to increase the stringency and accuracy of the analysis. Furthermore, a differential transcript usage (DTU) analysis was applied, attempting to discern mRNAs directly affected by NMD from indirect effects. Our results show that despite the existing individual differences, UPF1, SMG6 and SMG7 similarly affect the abundance of most mRNAs. Among the NMD-targeted genes, we found a significant enrichment of miRNA host genes, in addition to the already reported snoRNA host genes (Lykke-Andersen et al., Genes Dev 2014). Many non-coding RNAs also appear to be targeted by NMD, depending on the presence of open reading frames (ORFs) in their sequence, consistent with recent reports showing ribosome association of many supposedly non-coding transcripts (Ingolia et al., Cell Reports 2014). Furthermore, we also obtained evidence of transcription upstream of canonical start sites, which appears to be partially cleared by the NMD pathway, but to a lesser extent than has been reported for yeast (Malabat et al., eLife 2015)

A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activitie

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cell

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cells

Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors

Nonsense-mediated decay is well known by the lucid definition of being a RNA surveillance mechanism that ensures the speedy degradation of mRNAs containing premature translation termination codons. However, as we review here, NMD is far from being a simple quality control mechanism; it also regulates the stability of many wild-type transcripts. We summarise the abundance of research that has characterised each of the NMD factors and present a unified model for the recognition of NMD substrates. The contentious issue of how and where NMD occurs is also discussed, particularly with regard to P-bodies and SMG6-driven endonucleolytic degradation. In recent years, the discovery of additional functions played by several of the NMD factors has further complicated the picture. Therefore, we also review the reported roles of UPF1, SMG1 and SMG6 in other cellular processe

tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNASec however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNASec transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNASec is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNASec genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNASec outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNASec, in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNASec gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatid