TY - JOUR

T1 - Arrangements of nucleotides flanking the start codon in the IRES of the hepatitis C virus in the IRES binary complex with the human 40S ribosomal subunit

AU - Babaylova, Elena S.

AU - Graifer, Dmitri M.

AU - Malygin, Alexey A.

AU - Karpova, Galina G.

N1 - Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

PY - 2018/5/1

Y1 - 2018/5/1

N2 - Genomic RNA of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 5′-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position −3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position −3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes.

AB - Genomic RNA of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 5′-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position −3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position −3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes.

KW - 40S ribosomal mRNA binding site

KW - Human 40S ribosomal subunit

KW - Human ribosomal proteins

KW - IRES of hepatitis C virus

KW - Mammalian translation initiation

KW - Site-directed cross-linking

KW - P-SITE

KW - CONTACTS

KW - ENTRY SITE

KW - EUKARYOTIC TRANSLATION INITIATION

KW - MESSENGER-RNA

KW - DOMAIN-II

KW - HCV

KW - PROTEINS

KW - BINDING

KW - STEPS

KW - Nucleotides/genetics

KW - Humans

KW - Base Sequence

KW - Female

KW - Placenta/virology

KW - Hepacivirus/genetics

KW - Codon, Initiator/genetics

KW - Internal Ribosome Entry Sites/genetics

KW - Ribosome Subunits, Small, Eukaryotic/genetics

KW - Pregnancy

UR - http://www.scopus.com/inward/record.url?scp=85043343882&partnerID=8YFLogxK

U2 - 10.1016/j.biochi.2018.02.016

DO - 10.1016/j.biochi.2018.02.016

M3 - Article

C2 - 29501734

AN - SCOPUS:85043343882

VL - 148

SP - 72

EP - 79

JO - Biochimie

JF - Biochimie

SN - 0300-9084

ER -