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The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Inokuchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest regarding the content of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Translation of the downstream coding sequence of some mRNAs may be repressed by the upstream open reading frame (uORF) at their 5'-end. The mechanism underlying this uORF-mediated translational inhibition (uORF-MTI) is not fully understood in vivo. Recently, it was found that zebrafish Endouc or its human orthologue ENDOU (Endouc/ENDOU) plays a positive role in repressing the uORF-MTI of human CHOP (uORF chop -MTI) during stress by blocking its activity However, the repression of uORF chop -MTI assisted by an as-yet unidentified negative effector remains to be elucidated. Compared to the upregulated CHOP transcript, we herein report that the kepi (kinase-enhanced PP1 inhibitor) transcript was downregulated in the zebrafish embryos treated with both heat shock and hypoxia. Quantitative RT-PCR also revealed that the level of kepi mRNA was noticeably decreased in both heat-shock-treated and hypoxia-exposed embryos. When kepi mRNA was microinjected into the one-celled embryos from transgenic line huORFZ, the translation of downstream GFP reporter controlled by the uORF chop -MTI was reduced in the hypoxia-exposed embryos. In contrast, when kepi was knocked down by injection of antisense Morpholino oligonucleotide, the translation of downstream GFP reporter was induced and expressed in the brain and spinal cord of injected embryos in the absence of stress. During normal condition, overexpression of KEPI increased eIF2α phosphorylation, resulting in inducing the translation of uORF-tag mRNA, such as ATF4 and CHOP mRNAs. However, during stress condition, overexpression of KEPI decreased eIF2α phosphorylation, resulting in reducing the GFP reporter and CHOP proteins. This is the first report to demonstrate that KEPI plays a negative role in uORF chop - mediated translation during ER stress., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The review discusses the role that proteins interacting with the translation termination factors eRF1 and eRF3 play in the control of protein synthesis and prionization. These proteins interact not only with each other, but also with many other proteins involved in controlling the efficiency of translation termination, and associate translation termination with other cell processes. The termination of translation is directly related not only to translation re-initiation and ribosome recycling, but also to mRNA stability and protein quality control. This connection is ensured by the interaction of eRF1 and eRF3 with proteins participating in various cell metabolic processes, such as mRNA transport from the nucleus into the cytoplasm (Dbp5/DDX19 and Gle1), ribosome recycling (Rli1/ABCE1), mRNA degradation (Upf proteins), and translation initiation (Pab1/PABP). In addition to genetic control, there is epigenetic control of translation termination. This mechanism is associated with prion polymerization of the Sup35 protein to form the [PSI^(+)] prion. The maintenance of the [PSI^(+)] prion, like other yeast prions, requires the operation of a system of molecular chaperones and protein sorting factors. The review considers in detail the interaction of the translation termination factors with proteins involved in various cellular processes.

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood. Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and translational reprogramming. Similar effects on signaling pathways, including mTOR activation, the translatome and mouse cerebellar development were observed upon deletion of the NMD factor Upf2 . Our data reveal that these quality control pathways that function to mitigate errors at distinct steps in translation can evoke similar cellular responses., Competing Interests: MT, SA, JC, SA No competing interests declared, (© 2021, Terrey et al.)

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3'-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5' of the stop codon, six nucleotides 3' of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3'-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3'-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3'-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: A.J. is co-founder, director, and research consultant for PTC Therapeutics Inc.

Translation of problematic mRNA sequences induces ribosome stalling, triggering quality-control events, including ribosome rescue and nascent polypeptide degradation. To define the timing and regulation of these processes, we developed a SunTag-based reporter to monitor translation of a problematic sequence (poly[A]) in real time on single mRNAs. Although poly(A)-containing mRNAs undergo continuous translation over the timescale of minutes to hours, ribosome load is increased by ∼3-fold compared to a control, reflecting long queues of ribosomes extending far upstream of the stall. We monitor the resolution of these queues in real time and find that ribosome rescue is very slow compared to both elongation and termination. Modulation of pause strength, collision frequency, and the collision sensor ZNF598 reveals how the dynamics of ribosome collisions and their recognition facilitate selective targeting for quality control. Our results establish that slow clearance of stalled ribosomes allows cells to distinguish between transient and deleterious stalls., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Transcription termination is a critical stage for the production of legitimate mRNAs, and consequently functional proteins. However, the transcription machinery can ignore the stop signs and continue elongating beyond gene boundaries, invading downstream neighboring genes. Such phenomenon, designated transcription readthrough, can trigger the expression of pseudogenes usually silenced or lacking the proper regulatory signals. Due to the sequence similarity to parental genes, readthrough transcribed pseudogenes can regulate relevant protein-coding genes and impact biological functions. Here, we describe a computational pipeline that employs already existent bioinformatic tools to detect readthrough transcribed pseudogenes from expression profiles. We also unveil that combining strand-specific transcriptome data and epigenetic profiles can enhance and corroborate the results. By applying such approach to renal cancer biopsies, we show that pseudogenes can be readthrough transcribed as part of unspliced transcripts or processed RNA chimeras. Overall, our pipeline allows us to scrutinize transcriptome profiles to detect a diversity of readthrough events leading to expression of pseudogenes.

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD)., Competing Interests: Declaration of Interests A.J. is co-founder, director, and SAB chair of PTC Therapeutics Inc. B.R. is an employee of New England Biolabs., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Translation termination is a finishing step of protein biosynthesis. The significant role in this process belongs not only to protein factors of translation termination but also to the nearest nucleotide environment of stop codons. There are numerous descriptions of stop codons readthrough, which is due to specific nucleotide sequences behind them. However, represented data are segmental and don't explain the mechanism of the nucleotide context influence on translation termination. It is well known that stop codon UAA usage is preferential for A/T-rich genes, and UAG, UGA-for G/C-rich genes, which is related to an expression level of these genes. We investigated the connection between a frequency of nucleotides occurrence in 3' area of stop codons in the human genome and their influence on translation termination efficiency. We found that 3' context motif, which is cognate to the sequence of a stop codon, stimulates translation termination. At the same time, the nucleotide composition of 3' sequence that differs from stop codon, decreases translation termination efficiency.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a "leaky" stop codon context, which likely defines the basis of nonsense suppression.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in C9orf72 (C9-HRE). While RNA and dipeptide repeats produced by C9-HRE disrupt nucleocytoplasmic transport, the proteins that become redistributed remain unknown. Here, we utilized subcellular fractionation coupled with tandem mass spectrometry and identified 126 proteins, enriched for protein translation and RNA metabolism pathways, which collectively drive a shift toward a more cytosolic proteome in C9-HRE cells. Among these was eRF1, which regulates translation termination and nonsense-mediated decay (NMD). eRF1 accumulates within elaborate nuclear envelope invaginations in patient induced pluripotent stem cell (iPSC) neurons and postmortem tissue and mediates a protective shift from protein translation to NMD-dependent mRNA degradation. Overexpression of eRF1 and the NMD driver UPF1 ameliorate C9-HRE toxicity in vivo. Our findings provide a resource for proteome-wide nucleocytoplasmic alterations across neurodegeneration-associated repeat expansion mutations and highlight eRF1 and NMD as therapeutic targets in C9orf72-associated ALS and/or FTD., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Biallelic PTPRQ pathogenic variants have been previously reported as causative for autosomal recessive non-syndromic hearing loss. In 2018 the first heterozygous PTPRQ variant has been implicated in the development of autosomal dominant non-syndromic hearing loss (ADNSHL) in a German family. The study presented the only, so far known, PTPRQ pathogenic variant (c.6881G>A) in ADNSHL. It is located in the last PTPRQ coding exon and introduces a premature stop codon (p.Trp2294*)., Methods: A five-generation Polish family with ADNSHL was recruited for the study (n = 14). Thorough audiological, neurotological and imaging studies were carried out to precisely define the phenotype. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted for the proband. Family segregation analysis of the identified variants was performed using Sanger sequencing. Single nucleotide polymorphism array on DNA samples from the Polish and the original German family was used for genome-wide linkage analysis., Results: Combining clinical exome sequencing and family segregation analysis, we have identified the same (NM_001145026.2:c.6881G>A, NP_001138498.1:p.Trp2294*) PTPRQ alteration in the Polish ADNSHL family. Using genome-wide linkage analysis, we found that the studied family and the original German family derive from a common ancestor. Deep phenotyping of the affected individuals showed that in contrast to the recessive form, the PTPRQ-related ADNSHL is not associated with vestibular dysfunction. In both families ADNSHL was progressive, affected mainly high frequencies and had a variable age of onset., Conclusion: Our data provide the first confirmation of PTPRQ involvement in ADNSHL. The finding strongly reinforces the inclusion of PTPRQ to the small set of genes leading to both autosomal recessive and dominant hearing loss.

Efforts to develop treatments for diseases caused by nonsense mutations have focused on identification of small molecules that promote translational read-through of messenger RNAs (mRNAs) harboring nonsense stop codons to produce full-length proteins. However, to date, no small molecule read-through drug has received FDA approval, probably because of a lack of balance between efficacy and safety. Depletion of translation termination factors eukaryotic release factor ( eRF ) 1 and eRF3a in cells was shown to promote translational read-through of a luciferase reporter gene harboring a nonsense mutation. In this study, we identified antisense oligonucleotides (ASOs) targeting translation termination factors and determined if ASO-mediated depletion of these factors could be a potentially effective and safe therapeutic approach for diseases caused by nonsense mutations. We found that ASO-mediated reduction of either eRF1 or eRF3a to 30%-40% of normal levels in the mouse liver is well tolerated. Hemophilia mice that express a mutant allele of human coagulation factor IX (FIX) containing nonsense mutation R338X were treated with eRF1 - or eRF3a -ASO. We found that although eRF1 - or eRF3a -ASO alone only elicited a moderate read-through effect on hFIX-R338X mRNA, both worked in synergy with geneticin, a small molecule read-through drug, demonstrating significantly increased production of functional full-length hFIX protein to levels that would rescue disease phenotypes in these mice. Overall our results indicate that modulating the translation termination pathway in the liver by ASOs may provide a novel approach to improving the efficacy of small molecule read-through drugs to treat human genetic diseases caused by nonsense mutations.

Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examine this ribosome recycling role in vivo by ribosome profiling and reporter assays and find that loss of Hcr1 leads to reinitiation of translation in 3' UTRs, consistent with a defect in recycling. However, the defect appears to be in the recycling of the 60S subunit, rather than the 40S subunit, because reinitiation does not require an AUG codon and is suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 cannot compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggers greater expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation., (Published by Elsevier Inc.)

Nonsense mutations constitute ~10% of TP53 mutations in cancer. They introduce a premature termination codon that gives rise to truncated p53 protein with impaired function. The aminoglycoside G418 can induce TP53 premature termination codon readthrough and thus increase cellular levels of full-length protein. Small molecule phthalimide derivatives that can enhance the readthrough activity of G418 have also been described. To determine whether readthrough enhancers exist among drugs that are already approved for use in humans, we tested seven antimalarial drugs for readthrough of the common R213X TP53 nonsense mutation in HDQ-P1 breast cancer cells. Mefloquine induced no TP53 readthrough activity as a single agent but it strongly potentiated readthrough by G418. The two enantiomers composing pharmaceutical mefloquine potentiated readthrough to similar levels in HDQ-P1 cells and also in SW900, NCI-H1688 and HCC1937 cancer cells with different TP53 nonsense mutations. Exposure to G418 and mefloquine increased p53 phosphorylation at Ser15 and P21 transcript levels following DNA damage, indicating p53 produced via readthrough was functional. Mefloquine does not appear to enhance readthrough via lysosomotropic effects as it did not significantly affect lysosomal pH, the cellular levels of G418 or its distribution in organellar or cytosolic fractions. The availability of a readthrough enhancer that is already approved for use in humans should facilitate study of the therapeutic potential of TP53 readthrough in preclinical cancer models., Competing Interests: The authors have declared that no competing interests exist.

Background: Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein., Methods: To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts., Results: G418 treatment induced translational readthrough of CTNS W138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts., Conclusions: Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.

Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The major form of autosomal dominant polycystic kidney disease is caused by heterozygous mutations in PKD1 , the gene that encodes polycystin-1 (PC1). Unlike PKD1 genes in the mouse and most other mammals, human PKD1 is unusual in that it contains two long polypyrimidine tracts in introns 21 and 22 (2.5 kbp and 602 bp, respectively; 97% cytosine and thymine). Although these polypyrimidine tracts have been shown to form thermodynamically stable segments of triplex DNA that can cause DNA polymerase stalling and enhance the local mutation rate, the efficiency of transcription and splicing across these cytosine- and thymine-rich introns has been unexplored., Methods: We used RT-PCR and Western blotting (using an mAb to the N terminus) to probe splicing events over exons 20-24 in the mouse and human PKD1 genes as well as Nanopore sequencing to confirm the presence of multiple splice forms., Results: Analysis of PC1 indicates that humans, but not mice, have a smaller than expected protein product, which we call Trunc_PC1. The findings show that Trunc_PC1 is the protein product of abnormal differential splicing across introns 21 and 22 and that 28.8%-61.5% of PKD1 transcripts terminate early., Conclusions: The presence of polypyrimidine tracts decreases levels of full-length PKD1 mRNA from normal alleles. In heterozygous individuals, low levels of full-length PC1 may reduce polycystin signaling below a critical "cystogenic" threshold., (Copyright © 2018 by the American Society of Nephrology.)

Translation and mRNA degradation are intimately connected, yet the mechanisms that link them are not fully understood. Here, we studied these mechanisms in embryonic stem cells (ESCs). Transcripts showed a wide range of stabilities, which correlated with their relative translation levels and that did not change during early ESC differentiation. The protein DHH1 links translation to mRNA stability in yeast; however, loss of the mammalian homolog, DDX6, in ESCs did not disrupt the correlation across transcripts. Instead, the loss of DDX6 led to upregulated translation of microRNA targets, without concurrent changes in mRNA stability. The Ddx6 knockout cells were phenotypically and molecularly similar to cells lacking all microRNAs ( Dgcr8 knockout ESCs). These data show that the loss of DDX6 can separate the two canonical functions of microRNAs: translational repression and transcript destabilization. Furthermore, these data uncover a central role for translational repression independent of transcript destabilization in defining the downstream consequences of microRNA loss., Competing Interests: JF, TH, RB No competing interests declared, (© 2018, Freimer et al.)

Mycobacterium tuberculosis is a critical threat to human health due to the increased prevalence of rifampin resistance (RMP r ). Fitness defects have been observed in RMP r mutants with amino acid substitutions in the β subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi β-barrel (DPBB) domain of the β' subunit of RNAP. To identify factors contributing to the fitness defects observed in vivo , several in vitro RNA transcription assays were utilized to probe initiation, elongation, termination, and 3'-RNA hydrolysis with the wild-type and RMP r M. tuberculosis RNAPs. We found that the less prevalent RMP r mutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMP r mutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β' subunit., (Copyright © 2018 American Society for Microbiology.)

Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3'-terminus to investigate mutual arrangement of the main components of translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3'-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A model Post-Termination Complex (PoTC) used for the discovery of Ribosome Recycling Factor (RRF) was purified and characterized by cryo-electron microscopic analysis and biochemical methods. We established that the model PoTC has mostly one tRNA, at the P/E or P/P position, together with one mRNA. The structural studies were supported by the biochemical measurement of bound tRNA and mRNA. Using this substrate, we establish that the release of tRNA, release of mRNA and splitting of ribosomal subunits occur during the recycling reaction. Order of these events is tRNA release first followed by mRNA release and splitting almost simultaneously. Moreover, we demonstrate that IF3 is not involved in any of the recycling reactions but simply prevents the re-association of split ribosomal subunits. Our finding demonstrates that the important function of RRF includes the release of mRNA, which is often missed by the use of a short ORF with the Shine-Dalgarno sequence near the termination site.

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Termination efficiency of stop codons depends on the first 3' flanking (+4) base in bacteria and eukaryotes. In both Escherichia coli and Saccharomyces cerevisiae, termination read-through is reduced in the presence of +4U; however, the molecular mechanism underlying +4U function is poorly understood. Here, we perform comparative genomics analysis on 25 bacterial species (covering Actinobacteria, Bacteriodetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria, and Spirochaetae) with bioinformatics approaches to examine the influence of +4U in bacterial translation termination by contrasting highly- and lowly-expressed genes (HEGs and LEGs, respectively). We estimated gene expression using the recently formulated Index of Translation Elongation, I TE , and identified stop codon near-cognate transfer RNAs (tRNAs) from well-annotated genomes. We show that +4U was consistently overrepresented in UAA-ending HEGs relative to LEGs. The result is consistent with the interpretation that +4U enhances termination mainly for UAA. Usage of +4U decreases in GC-rich species where most stop codons are UGA and UAG, with few UAA-ending genes, which is expected if UAA usage in HEGs drives up +4U usage. In HEGs, +4U usage increases significantly with abundance of UAA nc_tRNAs (near-cognate tRNAs that decode codons differing from UAA by a single nucleotide), particularly those with a mismatch at the first stop codon site. UAA is always the preferred stop codon in HEGs, and our results suggest that UAAU is the most efficient translation termination signal in bacteria., (Copyright © 2017 Wei and Xia.)

Poly(A)-binding protein (PABP) is a major component of the messenger RNA-protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl-tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1-eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45-Sup35 (eRF1-eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45-Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Terminating transcription is a highly intricate process for mammalian protein-coding genes. First, the chromatin template slows down transcription at the gene end. Then, the transcript is cleaved at the poly(A) signal to release the messenger RNA. The remaining transcript is selectively unraveled and degraded. This induces critical conformational changes in the heart of the enzyme that trigger termination. Termination can also occur at variable positions along the gene and so prevent aberrant transcript formation or intentionally make different transcripts. These may form multiple messenger RNAs with altered regulatory properties or encode different proteins. Finally, termination can be perturbed to achieve particular cellular needs or blocked in cancer or virally infected cells. In such cases, failure to terminate transcription can spell disaster for the cell., (Copyright © 2016, American Association for the Advancement of Science.)

Pseudouridylation of messenger RNA emerges as an abundant modification involved in gene expression regulation. Pseudouridylation of stop codons in eukaryotic and bacterial cells results in stop-codon read through. The structural mechanism of this phenomenon is not known. Here we present a 3.1-Å crystal structure of Escherichia coli release factor 1 (RF1) bound to the 70S ribosome in response to the ΨAA codon. The structure reveals that recognition of a modified stop codon does not differ from that of a canonical stop codon. Our in vitro biochemical results support this finding by yielding nearly identical rates for peptide release from E. coli ribosomes programmed with pseudouridylated and canonical stop codons. The crystal structure also brings insight into E. coli RF1-specific interactions and suggests involvement of L27 in bacterial translation termination. Our results are consistent with a mechanism in which read through of a pseudouridylated stop codon in bacteria results from increased decoding by near-cognate tRNAs (miscoding) rather than from decreased efficiency of termination., (Copyright © 2016. Published by Elsevier Ltd.)

Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5' end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies.

Selenocysteine (Sec) is translated from the codon UGA, typically a termination signal. Codon duality extends the genetic code; however, the coexistence of two competing UGA-decoding mechanisms immediately compromises proteome fidelity. Selenium availability tunes the reassignment of UGA to Sec. We report a CRL2 ubiquitin ligase-mediated protein quality-control system that specifically eliminates truncated proteins that result from reassignment failures. Exposing the peptide immediately N-terminal to Sec, a CRL2 recognition degron, promotes protein degradation. Sec incorporation destroys the degron, protecting read-through proteins from detection by CRL2. Our findings reveal a coupling between directed translation termination and proteolysis-assisted protein quality control, as well as a cellular strategy to cope with fluctuations in organismal selenium intake., (Copyright © 2015, American Association for the Advancement of Science.)

Frontotemporal lobar degeneration is associated with cytoplasmic or nuclear deposition of the TAR DNA-binding protein 43 (TDP-43). Haploinsufficiency of progranulin (GRN) is a major genetic risk factor for frontotemporal lobar degeneration associated with TDP-43 deposition. Therefore, understanding the mechanisms that control cellular expression of GRN is required not only to understand disease etiology but also for the development of potential therapeutic strategies. We identified different GRN transcripts with short (38-93 nucleotides) or long (219 nucleotides) 5' UTRs and demonstrate a cellular mechanism that represses translation of GRN mRNAs with long 5' UTRs. The long 5' UTR of GRN mRNA contains an upstream open reading frame (uORF) that is absent in all shorter transcripts. Because such UTRs can be involved in translational control as well as in mRNA stability, we compared the expression of GRN in cells expressing cDNAs with and without 5' UTRs. This revealed a selective repression of GRN translation and a reduction of mRNA levels by the 219-nucleotide-long 5' UTR. The specific ability of this GRN 5' UTR to repress protein expression was further confirmed by its transfer to an independent reporter. Deletion analysis identified a short stretch between nucleotides 76 and 125 containing two start codons within one uORF that is required and sufficient for repression of protein expression. Mutagenesis of the two AUG codons within the uORF is sufficient to reduce translational repression. Therefore initiating ribosomes at the AUGs of the uORF fail to efficiently initiate translation at the start codon of GRN. In parallel the 5' UTR also affects mRNA stability; thus two independent mechanisms determine GRN expression via mRNA stability and translational efficiency., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.

Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.

Although most mRNA molecules derived from protein-coding genes are destined to be translated into functional polypeptides, some are eliminated by cellular quality control pathways that collectively perform the task of mRNA surveillance. In the nonsense-mediated decay (NMD) pathway premature translation termination promotes the recruitment of a set of factors that destabilize a targeted mRNA. The same factors also seem to have key roles in repressing the translation of the mRNA, dissociating its terminating ribosome and messenger ribonucleoproteins (mRNPs), promoting the degradation of its truncated polypeptide product and possibly even feeding back to the site of transcription to interfere with splicing of the primary transcript.

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Protein synthesis by ribosomes takes place on a linear substrate but at non-uniform speeds. Transient pausing of ribosomes can affect a variety of co-translational processes, including protein targeting and folding. These pauses are influenced by the sequence of the messenger RNA. Thus, redundancy in the genetic code allows the same protein to be translated at different rates. However, our knowledge of both the position and the mechanism of translational pausing in vivo is highly limited. Here we present a genome-wide analysis of translational pausing in bacteria by ribosome profiling--deep sequencing of ribosome-protected mRNA fragments. This approach enables the high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare transfer RNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine-Dalgarno-(SD)-like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome possessing an altered anti-SD sequence, we show that pausing is due to hybridization between the mRNA and 16S ribosomal RNA of the translating ribosome. In protein-coding sequences, internal SD sequences are disfavoured, which leads to biased usage, avoiding codons and codon pairs that resemble canonical SD sites. Our results indicate that internal SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes.

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease., (© 2012 médecine/sciences – Inserm / SRMS.)

Mimivirus and Megavirus are the best characterized representatives of an expanding new family of giant viruses infecting Acanthamoeba. Their most distinctive features, megabase-sized genomes carried in particles of size comparable to that of small bacteria, fill the gap between the viral and cellular worlds. These giant viruses are also uniquely equipped with genes coding for central components of the translation apparatus. The presence of those genes, thought to be hallmarks of cellular organisms, revived fundamental interrogations on the evolutionary origin of these viruses and the link they might have with the emergence of eukaryotes. In this work, we focused on the Mimivirus-encoded translation termination factor gene, the detailed primary structure of which was elucidated using computational and experimental approaches. We demonstrated that the translation of this protein proceeds through two internal stop codons via two distinct recoding events: a frameshift and a readthrough, the combined occurrence of which is unique to these viruses. Unexpectedly, the viral gene carries an autoregulatory mechanism exclusively encountered in bacterial termination factors, though the viral sequence is related to the eukaryotic/archaeal class-I release factors. This finding is a hint that the virally-encoded translation functions may not be strictly redundant with the one provided by the host. Lastly, the perplexing occurrence of a bacterial-like regulatory mechanism in a eukaryotic/archaeal homologous gene is yet another oddity brought about by the study of giant viruses., Competing Interests: The authors have declared that no competing interests exist.

Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.

Introduction: We investigated the putative candidate biomarkers of graft rejection in peripheral blood of intestinal transplant patients., Materials and Methods: Peripheral blood gene expression analysis was performed in intestinal transplant patients. The results were matched with concurrent graft biopsies using bioinformatics., Results: Peripheral blood samples (n=11), of 3 adult patients [transplant day (n=1), no rejection (n=1), minimal rejection (n=2), mild rejection (n=5) and severe rejection (n=2)] were collected. Bioinformatics: Enrichment Analysis: The three most affected pathways differentially expressed in rejection versus a pool of healthy volunteers were related to protein translation: translation initiation, translation elongation termination, and translation in mitochondria, with p-values for all rejection stages in all patients in the 10-4 to 10-18 range. No significant enrichment was observed for these categories in the day of transplant sample. In addition to translation, significant enrichment of several immune response categories was observed in rejection samples. Subsequent gene set enrichment analysis verified these results. The level of enrichment was very high (p-values of 10-5-10-60) and increased with the level of rejection in all patients. Genes significantly down-regulated in translation related gene sets included ribosomal proteins RPL13A, RP L22, RPS23, RPL13 and RPL10A, that could be used as potential biomarkers for future experiments., Conclusion: In this pilot study we found a list of genes (involved in translation) significantly downregulated in the peripheral blood of three intestinal transplant patients during rejection. These results will be verified in further studies with increased number of patients and with isolation of peripheral blood subpopulations.

A structure-activity relationship (SAR) study was carried out to identify novel, small molecular weight compounds which induce readthrough of premature termination codons. In particular, analogs of RTC13, 1, were evaluated. In addition, hypothesizing that these compounds exhibit their activity by binding to the ribosome, we prepared the hybrid analogs 13 containing pyrimidine bases and these also showed good readthrough activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The genetic encoding of synthetic or "non-natural" amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG "stop" triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We found that the synthesis of histone H3 N-terminal peptide (tail) in a reconstituted protein synthesis system yielded fragmented peptides along with the full-length product. With the combined use of MALDI-TOF analysis and peptidyl-tRNA hydrolase cleavage of the Flag tagged product species, we concluded that the fragments were generated by peptidyl-tRNA drop-off at specific sites and subsequent translation continuation. Using the histone H3 tail we also found that peptidyl-tRNA drop-off is strongly correlated with the amino acid context. We envision that the system described here would be useful as a model system for studying peptidyl-tRNA drop-off events., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)