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The human ribosomal protein eL29 binds in vivo to the cognate mRNA by interacting with its coding sequence, as revealed from in-cell cross-linking data. / Babaylova, Elena S.; Kolobova, Alena V.; Gopanenko, Alexander V. и др.
в: Biochimie, Том 177, 01.10.2020, стр. 68-77.Результаты исследований: Научные публикации в периодических изданиях › статья › Рецензирование
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TY - JOUR
T1 - The human ribosomal protein eL29 binds in vivo to the cognate mRNA by interacting with its coding sequence, as revealed from in-cell cross-linking data
AU - Babaylova, Elena S.
AU - Kolobova, Alena V.
AU - Gopanenko, Alexander V.
AU - Tupikin, Alexey E.
AU - Kabilov, Marsel R.
AU - Malygin, Alexey A.
AU - Karpova, Galina G.
N1 - Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
PY - 2020/10/1
Y1 - 2020/10/1
N2 - The balance of ribosomal proteins is important for the assembly of ribosomal subunits and cell viability. The synthesis of ribosomal proteins in a eukaryotic cell is controlled by various mechanisms, including autoregulation, which so far has been revealed for only a few of these proteins. We applied the photoactivatable 4-thiouridine-enhanced cross-linking and immunoprecipitation assay to HEK293T cells overproducing FLAG-labeled human ribosomal protein eL29 (eL29FLAG) to determine which RNAs other than rRNA interact with eL29. We demonstrated that eL29FLAG was incorporated into 60S subunits, and that ribosomes with those containing eL29FLAG were competent in translation. Analysis of the next generation sequencing data obtained from a DNA library derived from RNA fragments with covalently attached eL29FLAG peptide residues showed that the protein was cross-linked to the mRNA of the eL29-coding gene, which turned out to be its only major RNA target. The eL29FLAG cross-linking sites were located in the 3′ part of the mRNA coding sequence (CDS). A specific helix that mimics the eL29 binding site on 28S rRNA was proposed as a site that is recognized by the protein upon its binding to the cognate mRNA. In addition, it was found that both eL29FLAG mRNA and eL29 mRNA, unlike those of other ribosomal proteins, were co-immunoprecipitated with eL29FLAG from the ribosome-depleted cell lysate, and recombinant eL29 inhibited the translation of the eL29 mRNA CDS transcript in a cell-free system. All this suggests that human eL29 regulates its own synthesis via a feedback mechanism by binding to the cognate mRNA, preventing its translation.
AB - The balance of ribosomal proteins is important for the assembly of ribosomal subunits and cell viability. The synthesis of ribosomal proteins in a eukaryotic cell is controlled by various mechanisms, including autoregulation, which so far has been revealed for only a few of these proteins. We applied the photoactivatable 4-thiouridine-enhanced cross-linking and immunoprecipitation assay to HEK293T cells overproducing FLAG-labeled human ribosomal protein eL29 (eL29FLAG) to determine which RNAs other than rRNA interact with eL29. We demonstrated that eL29FLAG was incorporated into 60S subunits, and that ribosomes with those containing eL29FLAG were competent in translation. Analysis of the next generation sequencing data obtained from a DNA library derived from RNA fragments with covalently attached eL29FLAG peptide residues showed that the protein was cross-linked to the mRNA of the eL29-coding gene, which turned out to be its only major RNA target. The eL29FLAG cross-linking sites were located in the 3′ part of the mRNA coding sequence (CDS). A specific helix that mimics the eL29 binding site on 28S rRNA was proposed as a site that is recognized by the protein upon its binding to the cognate mRNA. In addition, it was found that both eL29FLAG mRNA and eL29 mRNA, unlike those of other ribosomal proteins, were co-immunoprecipitated with eL29FLAG from the ribosome-depleted cell lysate, and recombinant eL29 inhibited the translation of the eL29 mRNA CDS transcript in a cell-free system. All this suggests that human eL29 regulates its own synthesis via a feedback mechanism by binding to the cognate mRNA, preventing its translation.
KW - Feedback mechanism
KW - HEK293T cell Transfection
KW - Human ribosomal protein eL29
KW - In cell RNA-Protein cross-linking
KW - Protein binding to its own mRNA
KW - Regulation of gene expression
KW - NUCLEAR
KW - TRANSLATION
KW - IDENTIFICATION
KW - BIOGENESIS
KW - PATHWAY
KW - GENES
KW - S14
KW - SITES
KW - EXPRESSION
KW - SUBUNIT
UR - http://www.scopus.com/inward/record.url?scp=85089797847&partnerID=8YFLogxK
U2 - 10.1016/j.biochi.2020.07.019
DO - 10.1016/j.biochi.2020.07.019
M3 - Article
C2 - 32798643
AN - SCOPUS:85089797847
VL - 177
SP - 68
EP - 77
JO - Biochimie
JF - Biochimie
SN - 0300-9084
ER -
ID: 25302522