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Title: 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation

Abstract

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon–anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. In conclusion, our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Authors:
ORCiD logo [1];  [2]; ORCiD logo [3];  [2]; ORCiD logo [4];  [5];  [1];  [6];  [6]; ORCiD logo [7];  [2]; ORCiD logo [4]
  1. Stanford Univ. School of Medicine, Stanford, CA (United States); Stanford Univ., Stanford, CA (United States)
  2. Uppsala Univ., Uppsala (Sweden)
  3. SLAC National Accelerator Lab., Menlo Park, CA (United States)
  4. Stanford Univ. School of Medicine, Stanford, CA (United States)
  5. Stanford Univ. School of Medicine, Stanford, CA (United States); Auburn Univ., Auburn, AL (United States)
  6. Chaim Sheba Medical Center, Tel-Hashomer (Israel); Tel Aviv Univ., Tel Aviv (Israel)
  7. The Univ. of Chicago, Chicago, IL (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1460737
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
Nature Structural & Molecular Biology
Additional Journal Information:
Journal Volume: 25; Journal Issue: 3; Journal ID: ISSN 1545-9993
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Choi, Junhong, Indrisiunaite, Gabriele, DeMirci, Hasan, Ieong, Ka -Weng, Wang, Jinfan, Petrov, Alexey, Prabhakar, Arjun, Rechavi, Gideon, Dominissini, Dan, He, Chuan, Ehrenberg, Mans, and Puglisi, Joseph D. 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation. United States: N. p., 2018. Web. doi:10.1038/s41594-018-0030-z.
Choi, Junhong, Indrisiunaite, Gabriele, DeMirci, Hasan, Ieong, Ka -Weng, Wang, Jinfan, Petrov, Alexey, Prabhakar, Arjun, Rechavi, Gideon, Dominissini, Dan, He, Chuan, Ehrenberg, Mans, & Puglisi, Joseph D. 2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation. United States. doi:10.1038/s41594-018-0030-z.
Choi, Junhong, Indrisiunaite, Gabriele, DeMirci, Hasan, Ieong, Ka -Weng, Wang, Jinfan, Petrov, Alexey, Prabhakar, Arjun, Rechavi, Gideon, Dominissini, Dan, He, Chuan, Ehrenberg, Mans, and Puglisi, Joseph D. Mon . "2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation". United States. doi:10.1038/s41594-018-0030-z. https://www.osti.gov/servlets/purl/1460737.
@article{osti_1460737,
title = {2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation},
author = {Choi, Junhong and Indrisiunaite, Gabriele and DeMirci, Hasan and Ieong, Ka -Weng and Wang, Jinfan and Petrov, Alexey and Prabhakar, Arjun and Rechavi, Gideon and Dominissini, Dan and He, Chuan and Ehrenberg, Mans and Puglisi, Joseph D.},
abstractNote = {Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon–anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. In conclusion, our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.},
doi = {10.1038/s41594-018-0030-z},
journal = {Nature Structural & Molecular Biology},
number = 3,
volume = 25,
place = {United States},
year = {2018},
month = {2}
}

Journal Article:
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Cited by: 18 works
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Figures / Tables:

Fig. 1 Fig. 1: Multiple cognate tRNAs are rejected during decoding of the 2′-O-methylated codon. a, Single-molecule experimental schematics. Fluorescently labeled preinitiation complex (fluorophores are marked by a green burst (Cy3B), a red burst (Cy5) and a black circle (BHQ-2)) is tethered to the surface of the ZMW well via biotinylated mRNA,more » and necessary translation factors are delivered in the beginning of the signal acquisition. b, Expected sequence of events between translocation from the previous codon and the peptidyl transfer reaction. Both translocation and peptidyl transfer events are detected from the ribosomal intersubunit conformation change from the rotated state to the nonrotated state via change in the dye-quencher signal. Independently, tRNA binding events are detected from colocalized fluorescence from fluorescently labeled tRNA. c, Representative experimental trace at 5 mM Mg2+ with the first-base-modified codon (AmAA) shows a long stall between the translocation to the modified codon and its peptidyl transfer event. d, Quantification of stall on different modified lysine codons at different Mg2+ conditions. The stall duration was measured as described in c and was normalized to the nonrotated-state lifetimes for the unmodified codon, as specified in Supplementary Note 1. Number of molecules (n) = 147, 154, 77, 111, 94, 111, 120 and 108 from left to right; error bars were calculated from propagating s.e.m. from fitting the single-exponential distributions; asterisk (*) on AAmA codon at 5 mM Mg2+ condition indicates that out of all traces that showed translation leading to the modified codon, less than 5% showed a tRNA binding or ribosome-rotation event on the modified codon, hindering calculation of the normalized stall duration). Source data for d are available in Supplementary Dataset 3.« less

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    Works referencing / citing this record:

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