TY - JOUR

T1 - Exploring the interactions of short RNAs with the human 40S ribosomal subunit near the mRNA entry site by EPR spectroscopy

AU - Malygin, Alexey A.

AU - Krumkacheva, Olesya A.

AU - Graifer, Dmitri M.

AU - Timofeev, Ivan O.

AU - Ochkasova, Anastasia S.

AU - Meschaninova, Maria I.

AU - Venyaminova, Alya G.

AU - Fedin, Matvey V.

AU - Bowman, Michael

AU - Karpova, Galina G.

AU - Bagryanskaya, Elena G.

N1 - Funding Information: Russian Ministry of Science and Higher Education [14.W03.31.0034 (megagrant), in part]; SB RAS Somplex scientific program ‘Interdisciplinary Integration Studies’ for 2018−2020 [AAAA-A17-117112340082-0, 0309-2018-0010, AAAA-A17-117121890008-2]; Russian Ministry of Science and Higher Education (MSHE) under 5–100 Excellence Programme (in part); MSHE for access to the EPR equipment. Funding for open access charge: Russian Ministry of Science and Higher Education within Grant 14.W03.31.0034 (megagrant). Conflict of interest statement. None declared. 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 - 2019/12/16

Y1 - 2019/12/16

N2 - The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3′-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2′-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.

AB - The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3′-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2′-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.

UR - http://www.scopus.com/inward/record.url?scp=85076329269&partnerID=8YFLogxK

U2 - 10.1093/nar/gkz1039

DO - 10.1093/nar/gkz1039

M3 - Article

C2 - 31724718

AN - SCOPUS:85076329269

VL - 47

SP - 11850

EP - 11860

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 22

ER -