2′-O-methylation in mRNA disrupts tRNA decoding during translation elongation

Choi, Junhong; Indrisiunaite, Gabriele; DeMi̇rci̇, Hasan; Ieong, Ka Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D.

doi:10.1038/s41594-018-0030-z

Cited by 105 publications

(108 citation statements)

References 43 publications

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“…Analysis of 441 smFRET traces indicated three classes of translation events ( Fig 4E): Complete translation of ORF (54%), aborted translation after 4 th amino acid (G 4 ) without Lys-(Cy5)-tRNA Lys sampling (defined as tRNA binding longer than >100 millisecond 48 the A-site Lys codon (45%), and one that exhibited Lys-(Cy5)-tRNA Lys sampling in aborted translation (1%). Considering that a majority of arrested ribosomes exhibited a non-rotated-like conformational state without (>100ms lifetime) A-site tRNA sampling necessary for a processive elongation, we hypothesize that the ribosome is in a non-canonical structural state that cannot make a stable interaction among rRNA monitoring bases and codon-anticodon duplex necessary for further elongation 48 . Such state may be a result of different pathing of an mRNA as well as a nascent-peptide molecule within the ribosome, possibly similar to the previously observed interaction among the ErmCL nascent-peptide, the ribosome exit tunnel and the antibiotic erythromycin 49 .…”

Section: Mainmentioning

confidence: 99%

Short translational ramp determines efficiency of protein synthesis

Verma¹,

Choi

Cottrell³

et al. 2019

Preprint

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It is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence 1-4 . These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5' translational ramp, likely contributes to the efficiency of protein synthesis 5-9 . Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias 6,9 , the rate of translation initiation 10-15 , mRNA and ribosome structure 11,12,14,[16][17][18] , or retention of initiation factors during early elongation events 19 .Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4 th or 5 th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

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Section: Mainmentioning

confidence: 99%

Short translational ramp determines efficiency of protein synthesis

Verma¹,

Choi

Cottrell³

et al. 2019

Preprint

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“…[4][5][6][7][8][9][10] Modified nucleotides in tRNA, rRNAa nd mRNAd on ot only affect RNAp rocessing,t ransport and stability,b ut these residues also impact mRNAt ranslation. [11][12][13][14][15] Despite these important findings,d etails are scarce and disputed on the distribution and functions of many modified nucleotides in different transcriptomes.O nly recently,h igh-throughput sequencing (NGS) methods coupled to antibody-directed enrichment provided comprehensive maps of m 6 Aa nd m 1 A residues in different species. [16][17][18] Foro ther RNAm odifications,the use of specific chemical reagents allowed their highthroughput mapping.…”

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confidence: 99%

AlkAniline‐Seq: Profiling of m⁷G and m³C RNA Modifications at Single Nucleotide Resolution

et al. 2018

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RNA modifications play essential roles in gene expression regulation. Only seven out of >150 known RNA modifications are detectable transcriptome‐wide by deep sequencing. Here we describe a new principle of RNAseq library preparation, which relies on a chemistry based positive enrichment of reads in the resulting libraries, and therefore leads to unprecedented signal‐to‐noise ratios. The proposed approach eschews conventional RNA sequencing chemistry and rather exploits the generation of abasic sites and subsequent aniline cleavage. The newly generated 5′‐phosphates are used as unique entry for ligation of an adapter in library preparation. This positive selection, embodied in the AlkAniline‐Seq, enables a deep sequencing‐based technology for the simultaneous detection of 7‐methylguanosine (m7G) and 3‐methylcytidine (m3C) in RNA at single nucleotide resolution. As a proof‐of‐concept, we used AlkAniline‐Seq to comprehensively validate known m7G and m3C sites in bacterial, yeast, and human cytoplasmic and mitochondrial tRNAs and rRNAs, as well as for identifying previously unmapped positions.

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“…The entire function of 2'-O-methylation remained unclear, which was limited to a research tool. The previous study suggested that Nm seemed to involved in immune response via mRNA 5' cap Nm sites, mRNA alternative splicing mediated by intron branch point, maintaining the secondary structure of rRNA, and regulating protein translation by interfering tRNA-mRNA or mRNA-rRNA interactions [5,6,11,14,21]. Based on our preliminary data shown in this article, the roles of Nm sites in cells were much more prevalent than we expected.…”

Section: Discussionmentioning

confidence: 55%

A novel platform of RNA 2′-O-methylation high-throughput and site-specific quantification tools revealed its broad distribution on mRNA

Tang

et al. 2020

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Epigenetic modifications of RNA play a critical role in RNA structure and gene regulation. Ribose 2'-O-methylation (Nm) is one of the most common modifications among them, featured with one methyl group added to the 2' hydroxyl of the ribose moiety of RNA nucleosides. Although 2'-O-methylation was widely present in different type active RNAs, such as mRNAs, tRNAs, rRNAs, and even miRNAs, its biological functions and significance are still largely unknown due to the lack of accuracy and efficient identification screening tools. In this study, we first time established an enzyme-based high throughput Nm sites detection method with base resolution, which was named NJU-seq (Nm site Judge Universally Sequencing). We effectively identified Nm sites in human cultured cell lines, plants and animals with this new sequencing screening technique. Through this approach, we observed high abundance and complexity of Nm sties in cellular RNAs cross different species samples. Our study suggested that the function and regulation of Nm sites are more complicated than previous studies revealed and worth further mechanistic investigation.

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2′-O-methylation in mRNA disrupts tRNA decoding during translation elongation

Cited by 105 publications

References 43 publications

Short translational ramp determines efficiency of protein synthesis

Short translational ramp determines efficiency of protein synthesis

AlkAniline‐Seq: Profiling of m⁷G and m³C RNA Modifications at Single Nucleotide Resolution

A novel platform of RNA 2′-O-methylation high-throughput and site-specific quantification tools revealed its broad distribution on mRNA

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2′-O-methylation in mRNA disrupts tRNA decoding during translation elongation

Cited by 105 publications

References 43 publications

Short translational ramp determines efficiency of protein synthesis

Short translational ramp determines efficiency of protein synthesis

AlkAniline‐Seq: Profiling of m7G and m3C RNA Modifications at Single Nucleotide Resolution

A novel platform of RNA 2′-O-methylation high-throughput and site-specific quantification tools revealed its broad distribution on mRNA

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Product

Resources

About

AlkAniline‐Seq: Profiling of m⁷G and m³C RNA Modifications at Single Nucleotide Resolution