TY - JOUR

T1 - MRNA regions where 80S ribosomes pause during translation elongation in vivo interact with protein uS19, a component of the decoding site

AU - Babaylova, Elena S.

AU - Gopanenko, Alexander V.

AU - Bulygin, Konstantin N.

AU - Tupikin, Alexey E.

AU - Kabilov, Marsel R.

AU - Malygin, Alexey A.

AU - Karpova, Galina G.

N1 - Publisher Copyright: © 2019 The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.

PY - 2020/1/24

Y1 - 2020/1/24

N2 - In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.

AB - In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.

KW - Codon

KW - Eukaryota/genetics

KW - HEK293 Cells

KW - Humans

KW - Protein Biosynthesis/genetics

KW - RNA, Messenger/chemistry

KW - RNA, Transfer/chemistry

KW - Ribosomal Proteins/chemistry

KW - Ribosomes/chemistry

KW - Thiouridine/chemistry

UR - http://www.scopus.com/inward/record.url?scp=85077779986&partnerID=8YFLogxK

U2 - 10.1093/nar/gkz1145

DO - 10.1093/nar/gkz1145

M3 - Article

C2 - 31802126

AN - SCOPUS:85077779986

VL - 48

SP - 912

EP - 923

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 2

ER -