Cytosolic YB-1 and NSUN2 are the only proteins recognizing specific motifs present in mRNAs enriched in exosomes.
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.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
Olga A
OA
Kossinova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia; Department of Molecular Biology, Novosibirsk State University, Novosibirsk 630090, Russia.
Alexander V
AV
Gopanenko
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia; Department of Molecular Biology, Novosibirsk State University, Novosibirsk 630090, Russia.
Svetlana N
SN
Tamkovich
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
Olga A
OA
Krasheninina
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
Alexey E
AE
Tupikin
Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 10, Novosibirsk 630090, Russia.
Elena
E
Kiseleva
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
Darya D
DD
Yanshina
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia; Department of Molecular Biology, Novosibirsk State University, Novosibirsk 630090, Russia.
Alexey A
AA
Malygin
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
Alia G
AG
Ven'yaminova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
Marsel R
MR
Kabilov
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia; Department of Molecular Biology, Novosibirsk State University, Novosibirsk 630090, Russia. Electronic address: karpova@niboch.nsc.ru.
Galina G
GG
Karpova
D000595:Amino Acid Sequence; D003600:Cytosol / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051840:Y-Box-Binding Protein 1 / Q000737:chemistry / Q000378:metabolism*
D012333:RNA, Messenger; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D008780:Methyltransferases; C515610:NSUN2 protein, human
Exosomal RNA-specific motifs; NSUN2; Pull-down assay of cytosolic extract; RNA-protein interactions; YB-1; mRNAs species enriched in exosomes