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Firth, Andrew E., and Ian Brierley. "Non-canonical translation in RNA viruses." Journal of General Virology 93, no. 7 (2012): 1385–409. http://dx.doi.org/10.1099/vir.0.042499-0.
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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.
2
Prasad, Sharanya, Shelley Starck, and Nilabh Shastri. "Presentation of cryptic peptides by MHC I molecules is enhanced by inflammatory stimuli. (P5003)." Journal of Immunology 190, no. 1_Supplement (2013): 110.2. http://dx.doi.org/10.4049/jimmunol.190.supp.110.2.
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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.
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Coldwell, Mark J., Ulrike Sack, Joanne L. Cowan, et al. "Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon." Biochemical Journal 448, no. 1 (2012): 1–11. http://dx.doi.org/10.1042/bj20111765.
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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.
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Graça, Rafael, Rafael Fernandes, Ana Catarina Alves, Juliane Menezes, Luísa Romão, and Mafalda Bourbon. "Characterization of Two Variants at Met 1 of the Human LDLR Gene Encoding the Same Amino Acid but Causing Different Functional Phenotypes." Biomedicines 9, no. 9 (2021): 1219. http://dx.doi.org/10.3390/biomedicines9091219.
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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.
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Gao, Fei, Maria Wesolowska, Reuven Agami, et al. "Using mitoribosomal profiling to investigate human mitochondrial translation." Wellcome Open Research 2 (December 11, 2017): 116. http://dx.doi.org/10.12688/wellcomeopenres.13119.1.
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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.
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Gao, Fei, Maria Wesolowska, Reuven Agami, et al. "Using mitoribosomal profiling to investigate human mitochondrial translation." Wellcome Open Research 2 (January 29, 2018): 116. http://dx.doi.org/10.12688/wellcomeopenres.13119.2.
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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.
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Fecher-Trost, Claudia, Ulrich Wissenbach, Andreas Beck, et al. "The in Vivo TRPV6 Protein Starts at a Non-AUG Triplet, Decoded as Methionine, Upstream of Canonical Initiation at AUG." Journal of Biological Chemistry 288, no. 23 (2013): 16629–44. http://dx.doi.org/10.1074/jbc.m113.469726.
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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.
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Jewett, Mollie W., Sunny Jain, Angelika K. Linowski, Amit Sarkar, and Patricia A. Rosa. "Molecular characterization of the Borrelia burgdorferi in vivo-essential protein PncA." Microbiology 157, no. 10 (2011): 2831–40. http://dx.doi.org/10.1099/mic.0.051706-0.
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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.
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Paudel, Dinesh Babu, and Hélène Sanfaçon. "Mapping of sequences in the 5’ region and 3’ UTR of tomato ringspot virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro." PLOS ONE 16, no. 4 (2021): e0249928. http://dx.doi.org/10.1371/journal.pone.0249928.
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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.
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Alekhina, Olga, Ilya Terenin, Sergey Dmitriev, and Konstantin Vassilenko. "Functional Cyclization of Eukaryotic mRNAs." International Journal of Molecular Sciences 21, no. 5 (2020): 1677. http://dx.doi.org/10.3390/ijms21051677.
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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.
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Rahman, M. Sayeedur, Jason A. Simser, Kevin R. Macaluso, and Abdu F. Azad. "Functional analysis of secA homologues from rickettsiae." Microbiology 151, no. 2 (2005): 589–96. http://dx.doi.org/10.1099/mic.0.27556-0.
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The molecular basis of protein secretion that underlines rickettsial pathogenesis remains unknown. This paper reports the molecular and functional analysis of the putative secA gene, an essential component of the Sec-dependent protein secretion pathway, from Rickettsia rickettsii and Rickettsia typhi, the aetiological agents of Rocky Mountain spotted fever and murine typhus, respectively. The sequence analysis of the cloned secA genes from R. rickettsii and R. typhi show ORFs of 2721 and 2718 nt, respectively. Alignment of the deduced amino acid sequences reveals the presence of highly conserved amino acid residues and motifs considered to be essential for the ATPase activity of SecA in preprotein translocation. Transcription analysis indicates that R. rickettsii secA is expressed monocistronically from the canonical prokaryotic promoter, with a transcriptional start point located 32 nt upstream of the secA initiation codon. Complementation analysis shows that the full-length SecA protein from R. rickettsii and R. typhi fails to restore growth of the temperature-sensitive Escherichia coli strain MM52 secA51(ts) at a non-permissive temperature (42 °C), despite the detection of SecA protein expression by Western blotting. However, the chimeric SecA protein carrying the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA restores the growth of E. coli strain MM52 secA51(ts) at the non-permissive temperature (42 °C). These results suggest that the N-terminal ATPase domain is highly conserved, whereas the C-terminal domain appears to be species specific.
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Truniger, Verónica, Giuliano Sting Pechar, and Miguel A. Aranda. "Advances in Understanding the Mechanism of Cap-Independent Cucurbit Aphid-Borne Yellows Virus Protein Synthesis." International Journal of Molecular Sciences 24, no. 24 (2023): 17598. http://dx.doi.org/10.3390/ijms242417598.
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Non-canonical translation mechanisms have been described for many viral RNAs. In the case of several plant viruses, their protein synthesis is controlled by RNA elements in their genomic 3′-ends that are able to enhance cap-independent translation (3′-CITE). The proposed general mechanism of 3′-CITEs includes their binding to eukaryotic translation initiation factors (eIFs) that reach the 5′-end and AUG start codon through 5′-3′-UTR-interactions. It was previously shown that cucurbit aphid-borne yellows virus (CABYV) has a 3′-CITE, which varies in sequence and structure depending on the phylogenetic group to which the isolate belongs, possibly as a result of adaptation to the different geographical regions. In this work, the cap-independent translation mechanisms of two CABYV 3′-CITEs belonging to the Mediterranean (CMTE) and Asian (CXTE) groups, respectively, were studied. In vivo cap-independent translation assays show that these 3′-CITEs require the presence of the CABYV short genomic 5′-UTR with at least 40% adenines in cis and an accessible 5′-end for its activity. Additionally, they suggest that the eIF4E-independent CABYV 3′-CITE activities may not require either eIF4A or the eIF4F complex, but may depend on eIF4G and PABP. By pulling down host proteins using RNA baits containing both 5′- and 3′-CABYV-UTRs, 80 RNA binding proteins were identified. These interacted preferentially with either CMTE, CXTE, or both. One of these proteins, specifically interacting with the RNA containing CMTE, was HSP70.2. Preliminary results suggested that HSP70.2 may be involved in CMTE- but not CXTE-mediated cap-independent translation activity.
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Wu, Yu, Jianling Xie, Xin Jin, et al. "eEF2K enhances expression of PD-L1 by promoting the translation of its mRNA." Biochemical Journal 477, no. 22 (2020): 4367–81. http://dx.doi.org/10.1042/bcj20200697.
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Emerging advances in cancer therapy have transformed the landscape towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are ‘game-changers’ for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that negatively modulates the elongation stage of protein synthesis, promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5′-UTR (5′-untranslated region) which starts with a non-canonical CUG as the initiation codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5′-region. This, and other roles of eEF2K in cancer cell biology (e.g. in cell survival and migration), may be exploited for the design of future therapeutic strategies.
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Starck, Shelley R., Vivian Jiang, Mariana Pavon-Eternod, et al. "Leucine-tRNA Initiates at CUG Start Codons for Protein Synthesis and Presentation by MHC Class I." Science 336, no. 6089 (2012): 1719–23. http://dx.doi.org/10.1126/science.1220270.
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Effective immune surveillance by cytotoxic T cells requires newly synthesized polypeptides for presentation by major histocompatibility complex (MHC) class I molecules. These polypeptides are produced not only from conventional AUG-initiated, but also from cryptic non–AUG-initiated, reading frames by distinct translational mechanisms. Biochemical analysis of ribosomal initiation complexes at CUG versus AUG initiation codons revealed that cells use an elongator leucine-bound transfer RNA (Leu-tRNA) to initiate translation at cryptic CUG start codons. CUG/Leu-tRNA initiation was independent of the canonical initiator tRNA (AUG/Met-tRNAiMet) pathway but required expression of eukaryotic initiation factor 2A. Thus, a tRNA-based translation initiation mechanism allows non–AUG-initiated protein synthesis and supplies peptides for presentation by MHC class I molecules.
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Pan, Bingchen, Bowen Zheng, Chengzhong Xing, and Jingwei Liu. "Non-Canonical Programmed Cell Death in Colon Cancer." Cancers 14, no. 14 (2022): 3309. http://dx.doi.org/10.3390/cancers14143309.
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Programmed cell death (PCD) is an evolutionarily conserved process of cell suicide that is regulated by various genes and the interaction of multiple signal pathways. Non-canonical programmed cell death (PCD) represents different signaling excluding apoptosis. Colon cancer is the third most incident and the fourth most mortal worldwide. Multiple factors such as alcohol, obesity, and genetic and epigenetic alternations contribute to the carcinogenesis of colon cancer. In recent years, emerging evidence has suggested that diverse types of non-canonical programmed cell death are involved in the initiation and development of colon cancer, including mitotic catastrophe, ferroptosis, pyroptosis, necroptosis, parthanatos, oxeiptosis, NETosis, PANoptosis, and entosis. In this review, we summarized the association of different types of non-canonical PCD with tumorigenesis, progression, prevention, treatments, and prognosis of colon cancer. In addition, the prospect of drug-resistant colon cancer therapy related to non-canonical PCD, and the interaction between different types of non-canonical PCD, was systemically reviewed.
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Meinnel, T., C. Sacerdot, M. Graffe, S. Blanquet, and M. Springer. "Discrimination by Escherichia coli initiation factor IF3 against initiation on non-canonical codons relies on complementarity rules." Journal of Molecular Biology 290, no. 4 (1999): 825–37. http://dx.doi.org/10.1006/jmbi.1999.2881.
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Castelli, Lydia M., Wan-Ping Huang, Ya-Hui Lin, Kung-Yao Chang, and Guillaume M. Hautbergue. "Mechanisms of repeat-associated non-AUG translation in neurological microsatellite expansion disorders." Biochemical Society Transactions 49, no. 2 (2021): 775–92. http://dx.doi.org/10.1042/bst20200690.
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Repeat-associated non-AUG (RAN) translation was discovered in 2011 in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). This non-canonical form of translation occurs in all reading frames from both coding and non-coding regions of sense and antisense transcripts carrying expansions of trinucleotide to hexanucleotide repeat sequences. RAN translation has since been reported in 7 of the 53 known microsatellite expansion disorders which mainly present with neurodegenerative features. RAN translation leads to the biosynthesis of low-complexity polymeric repeat proteins with aggregating and cytotoxic properties. However, the molecular mechanisms and protein factors involved in assembling functional ribosomes in absence of canonical AUG start codons remain poorly characterised while secondary repeat RNA structures play key roles in initiating RAN translation. Here, we briefly review the repeat expansion disorders, their complex pathogenesis and the mechanisms of physiological translation initiation together with the known factors involved in RAN translation. Finally, we discuss research challenges surrounding the understanding of pathogenesis and future directions that may provide opportunities for the development of novel therapeutic approaches for this group of incurable neurodegenerative diseases.
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Monteuuis, Geoffray, Anna Miścicka, Michał Świrski, et al. "Non-canonical translation initiation in yeast generates a cryptic pool of mitochondrial proteins." Nucleic Acids Research 47, no. 11 (2019): 5777–91. http://dx.doi.org/10.1093/nar/gkz301.
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Abstract Utilization of non-AUG alternative translation start sites is most common in bacteria and viruses, but it has been also reported in other organisms. This phenomenon increases proteome complexity by allowing expression of multiple protein isoforms from a single gene. In Saccharomyces cerevisiae, a few described cases concern proteins that are translated from upstream near-cognate start codons as N-terminally extended variants that localize to mitochondria. Using bioinformatics tools, we provide compelling evidence that in yeast the potential for producing alternative protein isoforms by non-AUG translation initiation is much more prevalent than previously anticipated and may apply to as many as a few thousand proteins. Several hundreds of candidates are predicted to gain a mitochondrial targeting signal (MTS), generating an unrecognized pool of mitochondrial proteins. We confirmed mitochondrial localization of a subset of proteins previously not identified as mitochondrial, whose standard forms do not carry an MTS. Our data highlight the potential of non-canonical translation initiation in expanding the capacity of the mitochondrial proteome and possibly also other cellular features.
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Baudet, Mathieu, Philippe Ortet, Jean-Charles Gaillard, et al. "Proteomics-based Refinement ofDeinococcus desertiGenome Annotation Reveals an Unwonted Use of Non-canonical Translation Initiation Codons." Molecular & Cellular Proteomics 9, no. 2 (2009): 415–26. http://dx.doi.org/10.1074/mcp.m900359-mcp200.
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Schmitz, J. "Non-canonical translation mechanisms in plants: efficient in vitro and in planta initiation at AUU codons of the tobacco mosaic virus enhancer sequence." Nucleic Acids Research 24, no. 2 (1996): 257–63. http://dx.doi.org/10.1093/nar/24.2.257.
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Sikandar, Shaheen, Diana Dizon, Xiling Shen, Zuomei Li, Jeffery Besterman, and Steven M. Lipkin. "The Class I Hdac Inhibitor Mgcd0103 Induces Cell Cycle Arrest and Apoptosis in Colon Cancer Initiating Cells by Upregulating Dickkopf-1 and Non-Canonical Wnt Signaling." Oncotarget 1, no. 7 (2010): 596–605. http://dx.doi.org/10.18632/oncotarget.194.
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Slack, Jeffrey, Christopher Nguyen, and Amanda Ibe-Enwo. "A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios." Viruses 16, no. 1 (2023): 51. http://dx.doi.org/10.3390/v16010051.
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The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.
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Lin, Kangyu, and John Paul Shen. "Abstract 6066: Elucidating cancer stem cells heterogeneity in colorectal cancer by single-cell RNA sequencing." Cancer Research 82, no. 12_Supplement (2022): 6066. http://dx.doi.org/10.1158/1538-7445.am2022-6066.
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Abstract Cancer stem cells (CSCs) are a small subpopulation of cells with capabilities of self-renewal and differentiation potential and have been suggested to play a critical role in tumor-initiation, metastasis, and multidrug resistance. The marker genes used to identify CSCs in CRC and transcriptional state of these cell is not fully elucidated due to the plasticity and dynamic nature of CSCs. In this study, we perform single-cell RNA sequencing (scRNA-seq) on primary CRC and liver metastases from CRC to evaluate the CSC population. We profiled approximately 80,000 single cells from neoadjuvant chemotherapy (NAC) treated patients generated using 10X Chromium scRNA-seq and Illumina platforms. A total of 16 samples were analyzed (5 colon tumor, 3 colon matched normal, 3 liver metastasis, 5 liver matched normal), 4 samples failed quality control and were excluded. Following extensive quality control and batch effect correction, we performed dimensionality reduction and differential expression analysis with Seurat v3.0 and custom R packages. We used a set of 11 canonical colon CSC marker genes (CD44, PROM1, LGR5, LRIG1, ASCL2, EPHB2, OLFM4, AXIN2, SLC12A2, RNF43, LEFTY1) as CSCs signature to score the stemness of each cell by AddModuleScore. Established marker genes were used to identify cell type assignment including stem/TA, immature enterocytes, immature secretory, enterocytes, Best4 enterocytes, goblet, tuff, CD4, CD8, Treg, NK, B, plasma, macrophages, monocytes, dendritic cells, fibroblasts, endothelium, glia, pericytes and hepatocytes. In the 16,703 epithelial cells, the CSC signature genes were diffusely expressed in stem/TA and immature epithelium but not in a specific cell cluster. There was minimal overlap in expression of the 11 CSC marker genes at the single-cell level. By clustering the CSCs identified by high CSC signature score, we found CSCs grouped distinctly by patients, indicating significant patient to patient variability. Furthermore, CSCs did not cluster by positivity for any specific marker gene, suggesting that marker genes may just be markers, and do not define distinct transcriptional states. Gene set enrichment analysis (GSEA) showed intestinal stemness was significantly enriched (FDR < 0.01) in CSCs compared with non-CSCs. Comparing CSCs to stem cells from normal samples, suppression of the fatty acid metabolism pathway was the only common feature seen in CSCs from each tumor. The heterogeneity of CSC marker genes in CRC tumor cells suggests that defining CSCs by single or few marker genes is inadequate. GSEA verified that a CSC scoring system aggregating expression of 11 canonical colon CSC marker genes can identify CSCs from scRNA-seq. There is marked transcriptional heterogeneity in CSCs, with few common features from CSCs from different tumors. It is hoped that future study of how the diversity of CSCs states evolve under chemotherapy will shed light on overcoming chemo-resistance. Citation Format: Kangyu Lin, John Paul Shen. Elucidating cancer stem cells heterogeneity in colorectal cancer by single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6066.
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Price, Lauren E., Abigail B. Loewen Faul, Aleksandra Vuchkovska, et al. "Molecular Genetic Analysis of Rbm45/Drbp1: Genomic Structure, Expression, and Evolution." Journal of Student Research 7, no. 2 (2019): 49–61. http://dx.doi.org/10.47611/jsr.v7i2.426.
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RNA recognition motif-type RNA-binding domain containing proteins (RBDPs) participate in RNA metabolism including regulating mRNA stability, nuclear-cytoplasmic shuttling, and splicing. Rbm45 is an RBDP first cloned from rat brain and expressed spatiotemporally during rat neural development. More recently, RBM45 has been associated with pathological aggregates in the human neurological disorders amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer’s. Rbm45 and the neural developmental protein musashi-1 are in the same family of RDBPs and have similar expression patterns. In contrast to Musashi-1, which is upregulated during colorectal carcinogenesis, we found no association of RBM45 overexpression in human colon cancer tissue. In order to begin characterizing RNA-binding partners of Rbm45, we have successfully cloned and expressed human RBM45 in an Intein fusion-protein expression system. Furthermore, to gain a better understanding of the molecular genetics and evolution of Rbm45, we used an in silico approach to analyze the gene structure of the human and mouse Rbm45 homologues and explored the evolutionary conservation of Rbm45 in metazoans. Human RBM45 and mouse Rbm45 span ~17 kb and 13 kb, respectively, and contain 10 exons, one of which is non-coding. Both genes have TATA-less promoters with an initiator and a GC-rich element. Downstream of exon 10, both homologues have canonical polyadenylation signals and an embryonic cytoplasmic polyadenylation element. Moreover, our data indicate Rbm45 is conserved across all metazoan taxa from sponges (phylum Porifera) to humans (phylum Chordata), portending a fundamental role in metazoan development.
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Saks, Margaret E., John Oh, Austin C. Deets, George M. Mastorakos, and Susan Anne Martinis. "Translational Regulation of Gene Expression in Mycobacterium: A Means for Coordinating the Expression of Functionally Related Proteins." FASEB Journal 31, S1 (2017). http://dx.doi.org/10.1096/fasebj.31.1_supplement.759.7.
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Mycobacterium tuberculosis (Mtb) is one of the world's deadliest infectious agents. Consequently, a top priority has been to identify the mechanisms that Mtb uses to regulate the expression of genes involved in its virulence/persistence. Our over arching focus is on the translational regulation of gene expression. Our studies indicate that Mtb leverages differential codon usage and tRNA expression during both initiation and elongation to regulate protein synthesis.Translation initiation plays a key role in determining protein expression levels through its influence on the density of ribosomes bound on an mRNA. In bacteria, the initiation complex typically assembles with tRNAfMet bound to an AUG codon. However, sometimes the complex must assemble on a non‐canonical GUG or UUG initiation codon. In Mtb, and in the model organism M. smegmatis (Msm) which we use for our experiments, approximately forty percent of the initiation codons are non‐canonical. By contrast, non‐canonical initiation codons are used in only approximately 10 percent of the Escherichia coli coding sequences (CDS). We engineered an mCherry reporter system for use in Msm and used it to determine whether protein synthesis is differentially affected by initiation codon usage. Our experiments demonstrate that, throughout the growth curve, protein synthesis is substantially reduced when translation is initiated on GUG and UUG, rather than AUG codons. We are currently investigating whether canonical vs non‐canonical initiation codons are distributed across Mycobacterium coding sequences as a means for regulating protein synthesis. A conceptually related series of experiments focuses on the idea that, during elongation, the synthesis of functionally related proteins can be regulated though a “supply‐demand” mechanism. Due to the high GC content of the Mtb genome, the genome‐wide fraction of UUA (leucine) codons is only approximately1.6%. Interestingly the UUA codon is not randomly distributed across Mtb genes. Indeed, it is over‐represented in certain groups of genes involved in virulence and it is not used in the majority of Mtb coding sequences. These UUA‐containing genes can only be translated when the cognate tRNA, tRNALeuUAA is expressed. Therefore, the expression of functionally related proteins can be coordinated through the expression of tRNALeuUAA.Support or Funding InformationNational Institutes of Health GM63789
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Andreev, Dmitry E., Gary Loughran, Alla D. Fedorova, Maria S. Mikhaylova, Ivan N. Shatsky, and Pavel V. Baranov. "Non-AUG translation initiation in mammals." Genome Biology 23, no. 1 (2022). http://dx.doi.org/10.1186/s13059-022-02674-2.
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AbstractRecent proteogenomic studies revealed extensive translation outside of annotated protein coding regions, such as non-coding RNAs and untranslated regions of mRNAs. This non-canonical translation is largely due to start codon plurality within the same RNA. This plurality is often due to the failure of some scanning ribosomes to recognize potential start codons leading to initiation downstream—a process termed leaky scanning. Codons other than AUG (non-AUG) are particularly leaky due to their inefficiency. Here we discuss our current understanding of non-AUG initiation. We argue for a near-ubiquitous role of non-AUG initiation in shaping the dynamic composition of mammalian proteomes.
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Lee, Byeong Sung, Woon Jong Choi, Sang Woo Lee, Byoung Joon Ko, and Tae Hyeon Yoo. "Towards Engineering an Orthogonal Protein Translation Initiation System." Frontiers in Chemistry 9 (October 26, 2021). http://dx.doi.org/10.3389/fchem.2021.772648.
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In the last two decades, methods to incorporate non-canonical amino acids (ncAAs) into specific positions of a protein have advanced significantly; these methods have become general tools for engineering proteins. However, almost all these methods depend on the translation elongation process, and strategies leveraging the initiation process have rarely been reported. The incorporation of a ncAA specifically at the translation initiation site enables the installation of reactive groups for modification at the N-termini of proteins, which are attractive positions for introducing abiological groups with minimal structural perturbations. In this study, we attempted to engineer an orthogonal protein translation initiation system. Introduction of the identity elements of Escherichia coli initiator tRNA converted an engineered Methanococcus jannaschii tRNATyr into an initiator tRNA. The engineered tRNA enabled the site-specific incorporation of O-propargyl-l-tyrosine (OpgY) into the amber (TAG) codon at the translation initiation position but was inactive toward the elongational TAG codon. Misincorporation of Gln was detected, and the engineered system was demonstrated only with OpgY. We expect further engineering of the initiator tRNA for improved activity and specificity to generate an orthogonal translation initiation system.
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Ayyub, Shreya Ahana, Divya Dobriyal, and Umesh Varshney. "Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits." Journal of Bacteriology 199, no. 11 (2017). http://dx.doi.org/10.1128/jb.00051-17.
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ABSTRACT Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3. IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.
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Grove, Daisy J., Paul J. Russell, and Michael G. Kearse. "To initiate or not to initiate: A critical assessment of eIF2A, eIF2D, and MCT‐1·DENR to deliver initiator tRNA to ribosomes." WIREs RNA 15, no. 2 (2024). http://dx.doi.org/10.1002/wrna.1833.
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AbstractSelection of the correct start codon is critical for high‐fidelity protein synthesis. In eukaryotes, this is typically governed by a multitude of initiation factors (eIFs), including eIF2·GTP that directly delivers the initiator tRNA (Met‐tRNAiMet) to the P site of the ribosome. However, numerous reports, some dating back to the early 1970s, have described other initiation factors having high affinity for the initiator tRNA and the ability of delivering it to the ribosome, which has provided a foundation for further work demonstrating non‐canonical initiation mechanisms using alternative initiation factors. Here we provide a critical analysis of current understanding of eIF2A, eIF2D, and the MCT‐1·DENR dimer, the evidence surrounding their ability to initiate translation, their implications in human disease, and lay out important key questions for the field.This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes Translation > Mechanisms Translation > Regulation
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Miścicka, Anna, Kristen Lu, Irina S. Abaeva, Tatyana V. Pestova, and Christopher U. T. Hellen. "Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes." RNA, April 11, 2023, rna.079599.123. http://dx.doi.org/10.1261/rna.079599.123.
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Initiation of translation on many viral mRNAs occurs by non-canonical mechanisms that involve 5’ end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ~190 nt-long intergenic region (IGR) IRES of dicistroviruses such as Cricket paralysis virus (CrPV) initiates translation without Met-tRNAiMetor initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as Nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ~165 nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stemloop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) from which a CrPV-like stemloop SLIV and an adjacent conserved 5nt-long stemloop protrude.In vitroreconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAiMet. Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.
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Sonobe, Yoshifumi, Jihad Aburas, Gopinath Krishnan, et al. "A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation." Nature Communications 12, no. 1 (2021). http://dx.doi.org/10.1038/s41467-021-26303-x.
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AbstractA hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.
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Huntzinger, Eric, Jordan Sinteff, Bastien Morlet, and Bertrand Séraphin. "HELZ2: a new, interferon-regulated, human 3′-5′ exoribonuclease of the RNB family is expressed from a non-canonical initiation codon." Nucleic Acids Research, August 21, 2023. http://dx.doi.org/10.1093/nar/gkad673.
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Abstract Proteins containing a RNB domain, originally identified in Escherichia coli RNase II, are widely present throughout the tree of life. Many RNB proteins have 3′-5′ exoribonucleolytic activity but some have lost catalytic activity during evolution. Database searches identified a new RNB domain-containing protein in human: HELZ2. Analysis of genomic and expression data combined with evolutionary information suggested that the human HELZ2 protein is produced from an unforeseen non-canonical initiation codon in Hominidae. This unusual property was confirmed experimentally, extending the human protein by 247 residues. Human HELZ2 was further shown to be an active ribonuclease despite the substitution of a key residue in its catalytic center. HELZ2 RNase activity is lost in cells from some cancer patients as a result of somatic mutations. HELZ2 harbors also two RNA helicase domains and several zinc fingers and its expression is induced by interferon treatment. We demonstrate that HELZ2 is able to degrade structured RNAs through the coordinated ATP-dependent displacement of duplex RNA mediated by its RNA helicase domains and its 3′-5′ ribonucleolytic action. The expression characteristics and biochemical properties of HELZ2 support a role for this factor in response to viruses and/or mobile elements.
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Alghoul, Fatima, Schaeffer Laure, Gilbert Eriani, and Franck Martin. "Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition." eLife 10 (June 2, 2021). http://dx.doi.org/10.7554/elife.66369.
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During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The ‘start-stop’ sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.
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Zhang, Yanchao, Tom S. Bailey, Philip Hittmeyer, Ludwig J. Dubois, Jan Theys, and Philippe Lambin. "Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination." Microbial Cell Factories 23, no. 1 (2024). http://dx.doi.org/10.1186/s12934-024-02389-y.
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Abstract Background Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. Results Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. Conclusions This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.
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Singh, Jitendra, Rishi Kumar Mishra, Shreya Ahana Ayyub, Tanweer Hussain, and Umesh Varshney. "The initiation factor 3 (IF3) residues interacting with initiator tRNA elbow modulate the fidelity of translation initiation and growth fitness in Escherichia coli." Nucleic Acids Research, November 18, 2022. http://dx.doi.org/10.1093/nar/gkac1053.
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Abstract Initiation factor 3 (IF3) regulates the fidelity of bacterial translation initiation by debarring the use of non-canonical start codons or non-initiator tRNAs and prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain Escherichia coli growth. However, the roles of the N-terminal domain (NTD) have remained unclear. We hypothesized that the interaction between NTD and initiator tRNAfMet (i-tRNA) is essential to coordinate the movement of the two domains during the initiation pathway to ensure fidelity of the process. Here, using atomistic molecular dynamics (MD) simulation, we show that R25A/Q33A/R66A mutations do not impact NTD structure but disrupt its interaction with i-tRNA. These NTD residues modulate the fidelity of translation initiation and are crucial for bacterial growth. Our observations also implicate the role of these interactions in the subunit dissociation activity of CTD of IF3. Overall, the study shows that the interactions between NTD of IF3 and i-tRNA are crucial for coupling the movements of NTD and CTD of IF3 during the initiation pathway and in imparting growth fitness to E. coli.
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Sonobe, Yoshifumi, Soojin Lee, Gopinath Krishnan, et al. "Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons." eLife 12 (September 7, 2023). http://dx.doi.org/10.7554/elife.83189.
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A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide-repeat expansion disorders.
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Kienzle, Laura, Stefano Bettinazzi, Thierry Choquette, et al. "A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics." BMC Biology 21, no. 1 (2023). http://dx.doi.org/10.1186/s12915-023-01609-y.
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Abstract Background Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. Results We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. Conclusions Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.
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Rodriguez, Jose Manuel, Federico Abascal, Daniel Cerdán-Vélez, Laura Martínez Gómez, Jesús Vázquez, and Michael L. Tress. "Evidence for widespread translation of 5′ untranslated regions." Nucleic Acids Research, July 2, 2024. http://dx.doi.org/10.1093/nar/gkae571.
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Abstract Ribosome profiling experiments support the translation of a range of novel human open reading frames. By contrast, most peptides from large-scale proteomics experiments derive from just one source, 5′ untranslated regions. Across the human genome we find evidence for 192 translated upstream regions, most of which would produce protein isoforms with extended N-terminal ends. Almost all of these N-terminal extensions are from highly abundant genes, which suggests that the novel regions we detect are just the tip of the iceberg. These upstream regions have characteristics that are not typical of coding exons. Their GC-content is remarkably high, even higher than 5′ regions in other genes, and a large majority have non-canonical start codons. Although some novel upstream regions have cross-species conservation - five have orthologues in invertebrates for example - the reading frames of two thirds are not conserved beyond simians. These non-conserved regions also have no evidence of purifying selection, which suggests that much of this translation is not functional. In addition, non-conserved upstream regions have significantly more peptides in cancer cell lines than would be expected, a strong indication that an aberrant or noisy translation initiation process may play an important role in translation from upstream regions.