TY - JOUR

T1 - Cytosolic YB-1 and NSUN2 are the only proteins recognizing specific motifs present in mRNAs enriched in exosomes

AU - Kossinova, Olga A.

AU - Gopanenko, Alexander V.

AU - Tamkovich, Svetlana N.

AU - Krasheninina, Olga A.

AU - Tupikin, Alexey E.

AU - Kiseleva, Elena

AU - Yanshina, Darya D.

AU - Malygin, Alexey A.

AU - Ven'yaminova, Alia G.

AU - Kabilov, Marsel R.

AU - Karpova, Galina G.

N1 - Publisher Copyright: © 2017

PY - 2017/6/1

Y1 - 2017/6/1

N2 - Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs.

AB - Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs.

KW - Exosomal RNA-specific motifs

KW - mRNAs species enriched in exosomes

KW - NSUN2

KW - Pull-down assay of cytosolic extract

KW - RNA-protein interactions

KW - YB-1

KW - Amino Acid Sequence

KW - Humans

KW - Exosomes/metabolism

KW - Microscopy, Immunoelectron

KW - Flow Cytometry

KW - Methyltransferases/chemistry

KW - RNA, Messenger/chemistry

KW - Y-Box-Binding Protein 1/chemistry

KW - HEK293 Cells

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Cytosol/metabolism

UR - http://www.scopus.com/inward/record.url?scp=85016418559&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2017.03.010

DO - 10.1016/j.bbapap.2017.03.010

M3 - Article

C2 - 28341602

AN - SCOPUS:85016418559

VL - 1865

SP - 664

EP - 673

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 6

ER -