Nonstop Decay Research Articles

Saccharomyces cerevisiae Ski7 Is a GTP-Binding Protein Adopting the Characteristic Conformation of Active Translational GTPases

SummarySki7 is a cofactor of the cytoplasmic exosome in budding yeast, functioning in both mRNA turnover and non-stop decay (NSD), a surveillance pathway that degrades faulty mRNAs lacking a stop codon. The C-terminal region of Ski7 (Ski7C) shares overall sequence similarity with the translational GTPase (trGTPase) Hbs1, but whether Ski7 has retained the properties of a trGTPase is unclear. Here, we report the high-resolution structures of Ski7C bound to either intact guanosine triphosphate (GTP) or guanosine diphosphate-Pi. The individual domains of Ski7C adopt the conformation characteristic of active trGTPases. Furthermore, the nucleotide-binding site of Ski7C shares similar features compared with active trGTPases, notably the presence of a characteristic monovalent cation. However, a suboptimal polar residue at the putative catalytic site and an unusual polar residue that interacts with the γ-phosphate of GTP distinguish Ski7 from other trGTPases, suggesting it might function rather as a GTP-binding protein than as a GTP-hydrolyzing enzyme.

Ebola Virus GP Gene Polyadenylation Versus RNA Editing.

Synthesis of Ebola virus (EBOV) surface glycoprotein (GP) is dependent on transcriptional RNA editing. Northern blot analysis of EBOV-infected cells using GP-gene-specific probes reveals that, in addition to full-length GP messenger RNAs (mRNAs), a shorter RNA is also synthesized, representing >40% of the total amount of GP mRNA. Sequence analysis demonstrates that this RNA is a truncated version of the full-length GP mRNA that is polyadenylated at the editing site and thus lacks a stop codon. An absence of detectable levels of protein synthesis in cellulo is consistent with the existence of tight regulation of the translation of such mRNA. However, nonstop GP mRNA was shown to be only slightly less stable than the same mRNA containing a stop codon, against the general belief in nonstop decay mechanisms aimed at detecting and destroying mRNAs lacking a stop codon. In conclusion, we demonstrate that the editing site indeed serves as a cryptic transcription termination/polyadenylation site, which rarely also functions to edit GP mRNA for expression of surface GP. This new data suggest that the downregulation of surface GP expression is even more dramatic than previously thought, reinforcing the importance of the GP gene editing site for EBOV replication and pathogenicity.

Two degrading decades for RNA.

Two degrading decades for RNA.

Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems

The process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5’-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3’>5’direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals.

Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1

Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA(Arg(ICG)) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA(Arg(ICG)). Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA(Arg(ICG)) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA(Arg(ICG)). Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.

The Hbs1-Dom34 Protein Complex Functions in Non-stop mRNA Decay in Mammalian Cells

In yeast, aberrant mRNAs lacking in-frame termination codons are recognized and degraded by the non-stop decay (NSD) pathway. The recognition of non-stop mRNAs involves a member of the eRF3 family of GTP-binding proteins, Ski7. Ski7 is thought to bind the ribosome stalled at the 3'-end of the mRNA poly(A) tail and recruit the exosome to degrade the aberrant message. However, Ski7 is not found in mammalian cells, and even the presence of the NSD mechanism itself has remained enigmatic. Here, we show that unstable non-stop mRNA is degraded in a translation-dependent manner in mammalian cells. The decay requires another eRF3 family member (Hbs1), its binding partner Dom34, and components of the exosome-Ski complex (Ski2/Mtr4 and Dis3). Hbs1-Dom34 binds to form a complex with the exosome-Ski complex. Also, the elimination of aberrant proteins produced from non-stop transcripts requires the RING finger protein listerin. These findings demonstrate that the NSD mechanism exists in mammalian cells and involves Hbs1, Dom34, and the exosome-Ski complex.

MRNA surveillance is driven by translation

There are several mRNA surveillance pathways in eukaryotes that moderate the effects of natural errors in the cell and more broadly regulate gene expression. We have recently defined biochemical parameters of the no‐go decay quality control system using our previously developed in vitro reconstituted yeast translation system. In these studies, we have shown that two factors implicated in no‐go decay (NGD) and non‐stop decay (NSD), Dom34 and Hbs1, promote subunit dissociation and peptidyl‐tRNA drop‐off on the ribosome to initiate recycling events that eventually lead to mRNA decay. Of particular interest is that these factors (Dom34 and Hbs1) appear to identify stalled ribosome complexes that are associated with 3’ truncated mRNAs, suggesting a biochemical outcome for a likely in vivo consequence of stalling during translation (endonucleolytic cleavage of the mRNA). Current efforts focus on a broad analysis of the in vivo targets of mRNA surveillance (including NGD, NSD and NMD) using a series of reporter constructs and genome wide ribosome profiling.

The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro

The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.

Quality Control of Eukaryotic mRNA in The Process of Translation

Production of mature mRNA consists of a highly complex pathway of synthesis,and errors often happen.Both prokaryotic and eukaryotic cells have evolved remarkable surveillance mechanisms acting at several steps of mRNA biogenesis,even after translation initiation,to control mRNA quality.In eukaryotic cells,there are 4 translation-dependent mRNA surveillance pathways in cytoplasm,including nonsense-mediated decay(NMD),no-go decay(NGD),non-stop decay(NSD) and ribosome extension-mediated decay(REMD).These mRNA surveillance systems not only contribute to recognize and rapidly degrades aberrant mRNAs,but also play an essential role in gene regulation,and associated with several human diseases.In this review,recent achievements in the investigation of eukaryotic mRNA surveillance pathways will be discussed,and their application perspective will also be speculated.

Surveillance pathways rescuing eukaryotic ribosomes lost in translation

Living cells require the continuous production of proteins by the ribosomes. Any problem enforcing these protein factories to stall during mRNA translation may then have deleterious cellular effects. To minimize these defects, eukaryotic cells have evolved dedicated surveillance pathways: non-stop decay (NSD), no-go decay (NGD) and non-functional 18S-rRNA decay (18S-NRD). Recent studies support a general molecular framework for these surveillance pathways, the mechanisms of which are intimately related to translation termination.

Degradation of mRNAs that lack a stop codon: a decade of nonstop progress

Nonstop decay is the mechanism of identifying and disposing aberrant transcripts that lack in-frame stop codons. It is hypothesized that these transcripts are identified during translation when the ribosome arrives at the 3' end of the mRNA and stalls. Presumably, the ribosome stalling recruits additional cofactors, Ski7 and the exosome complex. The exosome degrades the transcript using either one of its ribonucleolytic activities, and the ribosome and the peptide are both released. Additional precautionary measures by the nonstop decay pathway may include translational repression of the nonstop transcript after translation, and proteolysis of the released peptide by the proteasome. This surveillance mechanism protects the cells from potentially harmful truncated proteins, but it may also be involved in mediating critical cellular functions of transcripts that are prone to stop codon read-through. Important advances have been made in the past decade as we learn that nonstop decay may have implications in human disease.

Translation drives mRNA quality control

There are three predominant forms of co-translational mRNA surveillance: nonsense-mediated decay (NMD), no-go decay (NGD) and nonstop decay (NSD). Although discussion of these pathways often focuses on mRNA fate, there is growing consensus that there are other important outcomes of these processes that must be simultaneously considered. Here, we seek to highlight similarities between NMD, NGD and NSD and their probable origins on the ribosome during translation.

Dom34:Hbs1 Plays a General Role in Quality-Control Systems by Dissociation of a Stalled Ribosome at the 3′ End of Aberrant mRNA

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.

Structural Basis for mRNA Surveillance by Archaeal Pelota and GTP-bound EF1α Complex

No-go decay (NGD) and Nonstop decay (NSD) are mRNA surveillance pathways that detect the stalled ribosome containing the defective mRNA. In archaea, archaeal Pelota (aPelota) associates with archaeal elongation factor 1α (aEF1α) to act in the mRNA surveillance pathway. Here, we present the complex structure of aPelota and GTP-bound aEF1α determined at 2.3 Å resolution. Notably, the aPelota·aEF1α·GTP complex structurally resembles the tRNA·EF-Tu complex bound to the ribosome. Combined with the functional analysis in yeast, our findings provide structural insights into how aPelota·aEF1α·GTP complex detects the stalled ribosome and triggers NGD and NSD.

MRNA stability and control of cell proliferation

Most of the studies on cell proliferation examine the control of gene expression by specific transcription factors that act on transcriptional initiation. In the last few years, it became evident that mRNA stability/turnover provides an important mechanism for post-transcriptional control of gene expression. In eukaryotes, mRNAs are mainly degraded after deadenylation by decapping and exosome pathways. Mechanisms of mRNA surveillance comprise deadenylation-independent pathways such as NMD (nonsense-mediated decay), when mRNAs harbour a PTC (premature termination codon), NSD (non-stop decay, when mRNAs lack a termination codon, and NGD (no-go decay), when mRNA translation elongation stalls. Many proteins involved in these processes are conserved from bacteria to yeast and humans. Recent papers showed the involvement of proteins deputed to decapping in controlling cell proliferation, virus replication and cell death. In this paper, we will review the newest findings in this field.

Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes

No-go decay (NGD) and non-stop decay (NSD) are eukaryotic surveillance mechanisms that target mRNAs on which elongation complexes (ECs) are stalled by, for example, stable secondary structures (NGD) or due to the absence of a stop codon (NSD). Two interacting proteins Dom34(yeast)/Pelota(mammals) and Hbs1, which are paralogues of eRF1 and eRF3, are implicated in these processes. Dom34/Hbs1 were shown to promote dissociation of stalled ECs and release of intact peptidyl-tRNA. Using an in vitro reconstitution approach, we investigated the activities of mammalian Pelota/Hbs1 and report that Pelota/Hbs1 also induced dissociation of ECs and release of peptidyl-tRNA, but only in the presence of ABCE1. Whereas Pelota and ABCE1 were essential, Hbs1 had a stimulatory effect. Importantly, ABCE1/Pelota/Hbs1 dissociated ECs containing only a limited number of mRNA nucleotides downstream of the P-site, which suggests that ABCE1/Pelota/Hbs1 would disassemble NSD complexes stalled at the 3'-end, but not pre-cleavage NGD complexes stalled in the middle of mRNA. ABCE1/Pelota/Hbs1 also dissociated vacant 80S ribosomes, which stimulated 48S complex formation, suggesting that Pelota/Hbs1 have an additional role outside of NGD.

Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs

Two general pathways of mRNA decay have been characterized in yeast. In one pathway, the mRNA is degraded by the cytoplasmic form of the exosome. The exosome has both 3' to 5' exoribonuclease and endoribonuclease activity, and the available evidence suggests that the exonuclease activity is required for the degradation of mRNAs. We confirm here that this is true for normal mRNAs, but that aberrant mRNAs that lack a stop codon can be efficiently degraded in the absence of the exonuclease activity of the exosome. Specifically, we show that the endo- and exonuclease activities of the exosome are both capable of rapidly degrading nonstop mRNAs and ribozyme-cleaved mRNAs. Additionally, the endonuclease activity of the exosome is not required for endonucleolytic cleavage in no-go decay. In vitro, the endonuclease domain of the exosome is active only under nonphysiological conditions, but our findings show that the in vivo activity is sufficient for the rapid degradation of nonstop mRNAs. Thus, whereas normal mRNAs are degraded by two exonucleases (Xrn1p and Rrp44p), several endonucleases contribute to the decay of many aberrant mRNAs, including transcripts subject to nonstop and no-go decay. Our findings suggest that the nuclease requirements for general and nonstop mRNA decay are different, and describe a molecular function of the core exosome that is not disrupted by inactivating its exonuclease activity.

Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1α complex

No-go decay and nonstop decay are mRNA surveillance pathways that detect translational stalling and degrade the underlying mRNA, allowing the correct translation of the genetic code. In eukaryotes, the protein complex of Pelota (yeast Dom34) and Hbs1 translational GTPase recognizes the stalled ribosome containing the defective mRNA. Recently, we found that archaeal Pelota (aPelota) associates with archaeal elongation factor 1α (aEF1α) to act in the mRNA surveillance pathway, which accounts for the lack of an Hbs1 ortholog in archaea. Here we present the complex structure of aPelota and GTP-bound aEF1α determined at 2.3-Å resolution. The structure reveals how GTP-bound aEF1α recognizes aPelota and how aPelota in turn stabilizes the GTP form of aEF1α. Combined with the functional analysis in yeast, the present results provide structural insights into the molecular interaction between eukaryotic Pelota and Hbs1. Strikingly, the aPelota·aEF1α complex structurally resembles the tRNA·EF-Tu complex bound to the ribosome. Our findings suggest that the molecular mimicry of tRNA in the distorted "A/T state" conformation by Pelota enables the complex to efficiently detect and enter the empty A site of the stalled ribosome.

Functional interactions between mRNA turnover and surveillance and the ubiquitin proteasome system

The proteasome is a critical regulator of protein levels within the cell and is essential for maintaining homeostasis. A functional proteasome is required for effective mRNA surveillance and turnover. During transcription, the proteasome localizes to sites of DNA breaks, degrading RNA polymerase II and terminating transcription. For fully transcribed and processed messages, cytoplasmic surveillance is initiated with the pioneer round of translation. The proteasome is recruited to messages bearing premature termination codons, which trigger nonsense-mediated decay (NMD), as well as messages lacking a termination codon, which trigger nonstop decay, to degrade the aberrant protein produced from these messages. A number of proteins involved in mRNA translation are regulated in part by proteasome-mediated decay, including the initiation factors eIF4G, eIF4E, and eIF3a, and the poly(A)-binding protein (PABP) interacting protein, Paip2. eIF4E-BP (4E-BP) is differentially regulated by the proteasome: truncated to generate a protein with higher eIF4B binding or completely degraded, depending on its phosphorylation status. Finally, a functional proteasome is required for AU-rich-element (ARE)-mediated decay but the specific role the proteasome plays is unclear. There is data indicating the proteasome can bind to AREs, act as an endonuclease, and degrade ARE-binding proteins. How these events interact with the 5'-to-3' and 3'-to-5' decay pathways is unclear at this time; however, data is provided indicating that proteasomes colocalize with Xrn1 and the exosome RNases Rrp44 and Rrp6 in untreated HeLa cells.

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3′-Terminal Exon

Alternative splicing of 3'-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3'-untranslated regions that regulate their fate and their expression. The Xenopus alpha-tropomyosin pre-mRNA possesses a composite internal/3'-terminal exon (exon 9A9') that is differentially processed depending on the embryonic tissue. Exon 9A9' is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3'-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9' as a 3'-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9' in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9' as a 3'-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3'-end processing of the alpha-tropomyosin pre-mRNA.