Letteratura scientifica selezionata sul tema "Non-canonical initiation codon"

Viral protein synthesis is completely dependent upon the translational machinery of the host cell. However, many RNA virus transcripts have marked structural differences from cellular mRNAs that preclude canonical translation initiation, such as the absence of a 5′ cap structure or the presence of highly structured 5′UTRs containing replication and/or packaging signals. Furthermore, whilst the great majority of cellular mRNAs are apparently monocistronic, RNA viruses must often express multiple proteins from their mRNAs. In addition, RNA viruses have very compact genomes and are under intense selective pressure to optimize usage of the available sequence space. Together, these features have driven the evolution of a plethora of non-canonical translational mechanisms in RNA viruses that help them to meet these challenges. Here, we review the mechanisms utilized by RNA viruses of eukaryotes, focusing on internal ribosome entry, leaky scanning, non-AUG initiation, ribosome shunting, reinitiation, ribosomal frameshifting and stop-codon readthrough. The review will highlight recently discovered examples of unusual translational strategies, besides revisiting some classical cases.

Abstract Cytolytic T cells eliminate infected cells by recognizing intracellular peptides presented by MHC class I molecules. The antigenic peptides are derived primarily from newly synthesized proteins including those produced by cryptic translation. Previous studies have shown that in addition to the canonical AUG codon, translation can be initiated at non-AUG codons . Furthermore, translation initiation at non-AUG codons such as CUG is mechanistically distinct from canonical translation initiation as it is resistant to protein synthesis inhibitors that cause global translation shutdown. Here, we show that Toll-like receptor (TLR) signaling pathways involved in pathogen recognition enhance presentation of the cryptic peptides. Moreover, infection of bone-marrow derived macrophages with MCMV, influenza viruses or pro-inflammatory cytokines also enhances cryptic peptide presentation. Thus, translation and presentation of cryptic peptides may allow the immune system to detect intracellular pathogens that inhibit host translation and presentation of peptides from conventional sources.

During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f–a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.

Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, characterized by increased levels of total and LDL plasma cholesterol, which leads to premature atherosclerosis and coronary heart disease. FH phenotype has considerable genetic heterogeneity and phenotypic variability, depending on LDL receptor activity and lifestyle. To improve diagnosis and patient management, here, we characterized two single nucleotide missense substitutions at Methionine 1 of the human LDLR gene (c.1A>T/p.(Met1Leu) and c.1A>C/p.(Met1Leu)). We used a combination of Western blot, flow cytometry, and luciferase assays to determine the effects of both variants on the expression, activity, and synthesis of LDLR. Our data show that both variants can mediate translation initiation, although the expression of variant c.1A>T is very low. Both variants are in the translation initiation codon and codify for the same amino acid p.(Met1Leu), yet they lead to different levels of impairment on LDLR expression and activity, corroborating different efficiencies of the translation initiation at these non-canonical initiation codons. The functional data of these variants allowed for an improved American College of Medical Genetics (ACMG) classification for both variants, which can allow a more personalized choice of the lipid-lowering treatment and dyslipidemia management, ultimately improving patients’ prognosis.

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNAPhe whilst in humans it is mt-tRNAVal. We have previously shown that when a mutation in mt-tRNAVal causes very low steady state levels, there is preferential recruitment of mt-tRNAPhe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNAPhe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNAPhe containing mitoribosomes with those of controls that integrate mt-tRNAVal revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNAVal cell line suggest that despite mt-tRNAPhe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNAPhe whilst in humans it is mt-tRNAVal. We have previously shown that when a mutation in mt-tRNAVal causes very low steady state levels, there is preferential recruitment of mt-tRNAPhe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNAPhe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNAPhe containing mitoribosomes with those of controls that integrate mt-tRNAVal revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNAVal cell line suggest that despite mt-tRNAPhe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

TRPV6 channels function as epithelial Ca2+ entry pathways in the epididymis, prostate, and placenta. However, the identity of the endogenous TRPV6 protein relies on predicted gene coding regions and is only known to a certain level of approximation. We show that in vivo the TRPV6 protein has an extended N terminus. Translation initiates at a non-AUG codon, at ACG, which is decoded by methionine and which is upstream of the annotated AUG, which is not used for initiation. The in vitro properties of channels formed by the extended full-length TRPV6 proteins and the so-far annotated and smaller TRPV6 are similar, but the extended N terminus increases trafficking to the plasma membrane and represents an additional scaffold for channel assembly. The increased translation of the smaller TRPV6 cDNA version may overestimate the in vivo situation where translation efficiency may represent an additional mechanism to tightly control the TRPV6-mediated Ca2+ entry to prevent deleterious Ca2+ overload.

The conversion of nicotinamide to nicotinic acid by nicotinamidase enzymes is a critical step in maintaining NAD+ homeostasis and contributes to numerous important biological processes in diverse organisms. In Borrelia burgdorferi, the nicotinamidase enzyme, PncA, is required for spirochaete survival throughout the infectious cycle. Mammals lack nicotinamidases and therefore PncA may serve as a therapeutic target for Lyme disease. Contrary to the in vivo importance of PncA, the current annotation for the pncA ORF suggests that the encoded protein may be inactive due to the absence of an N-terminal aspartic acid residue that is a conserved member of the catalytic triad of characterized PncA proteins. Herein, we have used genetic and biochemical strategies to determine the N-terminal sequence of B. burgdorferi PncA. Our data demonstrate that the PncA protein is 24 aa longer than the currently annotated sequence and that pncA translation is initiated from the rare, non-canonical initiation codon AUU. These findings are an important first step in understanding the catalytic function of this in vivo-essential protein.

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5’-viral genome-linked protein (VPg) and have a 3’ polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3’ untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5’ regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5’ region and 3’ UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5’ region and 3’ UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5’ proximal coding region of the RNA2 polyprotein and in the RNA2 3’ UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3’ UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3’ UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5’ region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773–972) of the 1547 nt long 3’ UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

Endant l’infection virale, la régulation de l’expression des gènes est au cœur des interactions complexes entre l'hôte et le pathogène. Les virus exploitent la machinerie cellulaire de l'hôte pour assurer la synthèse de leurs protéines nécessaires pour la réplication et la propagation de l'infection. C'est notamment le cas lors de l'infection par le SARS-CoV-2, qui induit rapidement une inhibition globale de la traduction cellulaire grâce à l'action de facteurs viraux tels que la protéine Nsp1. Pour produire efficacement ses protéines, le virus doit alors mettre en place des stratégies pour contourner cette inhibition. Le génome du SARS-CoV-2 s'exprime à partir de 10 ARN, l'ARN génomique (ARNg) et 9 ARN sous-génomiques qui possèdent une région leader commune mais des régions 5'UTR uniques pour chacun des transcrits. Mon travail s'est concentré sur les éléments structuraux qui régulent la traduction des différents ARN du SARS-CoV-2.À travers un ensemble d’expériences in vitro (lysat de réticulocytes) et en cellules, nous avons découvert que l’efficacité de traduction variait significativement entre les différents ARN viraux. En particulier, l'ARN génomique, malgré sa structure complexe, se distingue par une efficacité de traduction particulièrement élevée. Nous avons aussi déterminé que la structure tige-boucle SL1, présente dans l’ensemble des transcrits viraux, était un déterminant majeur pour l'expression des ARN et qu'elle jouait également un rôle crucial pour contrer l'inhibition induite par la protéine virale Nsp1. Nous avons établi que l'initiation de la traduction se déroulait par un mécanisme dépendant de la coiffe et nécessitait le complexe eIF4F. Enfin notre étude a également permis de caractériser le rôle de deux courtes phases de lecture ouvertes (uORF) retrouvées dans certaines régions 5'UTR des ARN du SARS-CoV-2; ces uORFs ont des impacts variables selon leur position
During viral infection, the regulation of gene expression is central to the complex interactions between the host and the pathogen. Viruses exploit the host's cellular machinery to ensure the synthesis of their proteins, which are necessary for replication and the spread of the infection. This is particularly the case with SARS-CoV-2 infection, which rapidly induces a global inhibition of cellular translation through the action of viral factors such as the Nsp1 protein. To efficiently produce its proteins, the virus must implement strategies to bypass this inhibition. The SARS-CoV-2 genome is expressed from 10 RNAs, the genomic RNA (gRNA) and 9 subgenomic RNAs that possess a common leader region but unique 5'UTR regions for each of the transcripts. My work focused on the structural elements that regulate the translation of the different SARS-CoV-2 RNAs.Through a series of in vitro (reticulocyte lysate) and in-cell experiments, we discovered that the translation efficiency varied significantly among the different viral RNAs. In particular, the genomic RNA, despite its complex structure, distinguishes itself by its remarkably high translation efficiency. We also determined that the SL1 stem-loop structure, present in all viral transcripts, was a major determinant for RNA expression and also played a crucial role in countering the inhibition induced by the Nsp1 viral protein. We established that translation initiation occurred through a cap-dependent mechanism and required the eIF4F complex. Finally, our study also characterized the role of two short upstream open reading frames (uORFs) found in certain 5'UTR regions of SARS-CoV-2 RNAs; these uORFs have variable impacts depending on their position

Alternative translation initiation from upstream non-AUG codons contributes towards the diversity of the eukaryotic proteome; protein isoforms with varying N-terminal extensions can be generated from one mRNA transcript. Investigations are being carried out in order to elucidate how N-terminal extensions affect subcellular localisation, binding partners and thus the function of a protein as well as how the choice of alternative initiation codon (AIC) is regulated. This thesis is focussed on GATAD1 (GATA Zinc Finger Domain-Containing 1), which is a ubiquitously expressed transcription factor, forming part of a transcriptionally repressive histone demethylase complex. GATAD1 regulates the expression of specific genes by forming an indirect interaction with the activating trimethyl marker of lysine four on histone three (H3K4me3); this interaction is made through a lysine demethylase chromatin ‘reader’, KDM5A (Jarid1A). GATAD1 is also involved in retinal development and heart disease, whereby a single mutation in the gene is the cause of dilated cardiomyopathy (DCM). The GATAD1 mRNA transcript can be translated at alternative translation initiation codons resulting in the synthesis of three protein isoforms. The two isoforms with N-terminal extensions are initiated from a CUG and an unusual AUU codon. CRISPR genome editing has been used to tag genomic GATAD1, confirming endogenous expression of all three isoforms. Translation from the AICs is regulated by various factors, including eukaryotic initiation factors (eIFs) 1, 1A and 5, the context and position of the AICs, as well as secondary structure downstream of the AUU codon. Cell type and stresses such as hypoxia also influence the use of each GATAD1 AIC. Although all three GATAD1 isoforms complex with KDM5A, it has been observed that the extended isoforms have a greater tendency to remain in the cytoplasm, potentially forming part of a cytoplasmic demethylase complex, whilst the annotated protein functions as a nuclear transcription factor.