TY - JOUR

T1 - Structural features of the interaction of the 3′-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting

AU - Yanshina, Darya D.

AU - Kossinova, Olga A.

AU - Gopanenko, Alexander V.

AU - Krasheninina, Olga A.

AU - Malygin, Alexey A.

AU - Venyaminova, Alya G.

AU - Karpova, Galina G.

N1 - Publisher Copyright: © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)

PY - 2018/1/1

Y1 - 2018/1/1

N2 - We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3′-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1–3 in the HEK293 cell mRNA 3′ UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3′ UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.

AB - We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3′-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1–3 in the HEK293 cell mRNA 3′ UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3′ UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.

KW - Exosomal RNA-specific motifs

KW - Pull-down assay of HEK293 cell cytosolic extract

KW - RNA footprinting

KW - RNA-protein interactions

KW - YB-1

KW - RIBONUCLEOPROTEIN PARTICLES

KW - INITIATION

KW - COMPLEXES

KW - RIBOSOMAL-RNA

KW - TRANSLATION

KW - P53

KW - BOX-BINDING-PROTEIN

KW - GENE-EXPRESSION

KW - SELECTION

KW - Humans

KW - Exosomes/metabolism

KW - Nucleotide Motifs

KW - Base Sequence

KW - HEK293 Cells

KW - Protein Binding

KW - 3' Untranslated Regions

KW - Binding Sites

KW - Y-Box-Binding Protein 1/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85034026196&partnerID=8YFLogxK

U2 - 10.1016/j.biochi.2017.11.007

DO - 10.1016/j.biochi.2017.11.007

M3 - Article

C2 - 29133115

AN - SCOPUS:85034026196

VL - 144

SP - 134

EP - 143

JO - Biochimie

JF - Biochimie

SN - 0300-9084

ER -