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      Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications

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          ABSTRACT

          Recently, a role for the anticodon wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine (mcm 5s 2U) has been revealed in the suppression of translational +1 frameshifts in Saccharomyces cerevisiae. Loss of either the mcm 5U or s 2U parts of the modification elevated +1 frameshift rates and results obtained with reporters involving a tRNA Lys UUU dependent frameshift site suggested these effects are caused by reduced ribosomal A-site binding of the hypomodified tRNA. Combined loss of mcm 5U and s 2U leads to increased ribosome pausing at tRNA Lys UUU dependent codons and synergistic growth defects but effects on +1 frameshift rates remained undefined to this end. We show in here that simultaneous removal of mcm 5U and s 2U results in synergistically increased +1 frameshift rates that are suppressible by extra copies of tRNA Lys UUU. Thus, two distinct chemical modifications of the same wobble base independently contribute to reading frame maintenance, loss of which may cause or contribute to observed growth defects. Since the thiolation pathway is sensitive to moderately elevated temperatures in yeast, we observe a heat-induced increase of +1 frameshift rates in wild type cells that depends on the sulfur transfer protein Urm1. Furthermore, we find that temperature-induced frameshifting is kept in check by the dehydration of N6-threonylcarbamoyladenosine (t 6A) to its cyclic derivative (ct 6A) at the anticodon adjacent position 37. Since loss of ct 6A in elp3 or urm1 mutant cells is detrimental for temperature stress resistance we assume that conversion of t 6A to ct 6A serves to limit deleterious effects on translational fidelity caused by hypomodified states of wobble uridine bases.

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          Most cited references42

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          The genetic landscape of a cell.

          A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.
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            Optimization of Codon Translation Rates via tRNA Modifications Maintains Proteome Integrity

            Summary Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress, accumulate aggregates of endogenous proteins, and are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA modifications in maintaining proteome integrity.
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              Biosynthesis and function of posttranscriptional modifications of transfer RNAs.

              Posttranscriptional modifications of transfer RNAs (tRNAs) are critical for all core aspects of tRNA function, such as folding, stability, and decoding. Most tRNA modifications were discovered in the 1970s; however, the near-complete description of the genes required to introduce the full set of modifications in both yeast and Escherichia coli is very recent. This led to a new appreciation of the key roles of tRNA modifications and tRNA modification enzymes as checkpoints for tRNA integrity and for integrating translation with other cellular functions such as transcription, primary metabolism, and stress resistance. A global survey of tRNA modification enzymes shows that the functional constraints that drive the presence of modifications are often conserved, but the solutions used to fulfill these constraints differ among different kingdoms, organisms, and species.
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                Author and article information

                Journal
                RNA Biol
                RNA Biol
                KRNB
                krnb20
                RNA Biology
                Taylor & Francis
                1547-6286
                1555-8584
                2017
                12 December 2016
                12 December 2016
                : 14
                : 9
                : 1252-1259
                Affiliations
                Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel , Kassel, Germany
                Author notes
                CONTACT Roland Klassen roland.klassen@ 123456uni-kassel.de Raffael Schaffrath schaffrath@ 123456uni-kassel.de Institut für Biologie, Universität Kassel, Fachgebiet Mikrobiologie , Heinrich-Plett-Str. 40, Kassel, Germany, D-34132
                Author information
                https://orcid.org/0000-0001-9484-5247
                Article
                1267098
                10.1080/15476286.2016.1267098
                5699549
                27937809
                945418f9-0e57-4cd7-a10b-33266ca9fc94
                © 2017 The Author(s). Published with license by Taylor & Francis Group, LLC

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

                History
                : 12 September 2016
                : 4 November 2016
                : 27 November 2016
                Page count
                Figures: 4, Tables: 2, Equations: 0, References: 44, Pages: 8
                Categories
                Research Paper

                Molecular biology
                5-methoxycarbonylmethyl-2-thiouridine,cyclic n6-threonylcarbamoyladenosine,trna modification, translation, translational frameshift

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