Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia.
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.
Biochim Biophys Acta Proteins Proteom. 2017 Jun;1865(6):664-673. doi: 10.1016/j.bbapap.2017.03.010. Epub 2017 Mar 21.
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.
外泌体是各种细胞分泌的膜性囊泡，参与细胞间通讯，携带大量 RNA 和蛋白质。目前，介导 RNA 分选到外泌体的过程还知之甚少。利用生物信息学方法，在人胚肾 293（HEK293）细胞胞质提取物的下拉实验中，发现三个结构基序 ACCAGCCU、CAGUGAGC 和 UAAUCCCA 在富含外泌体 mRNA 和长非编码 RNA 中富集。在这里，我们利用包含一个基序的短 RNA 发夹，在人胚肾 293（HEK293）细胞胞质提取物的下拉实验中，证明多功能 RNA 结合蛋白 YB-1 特异性地与这三个基序相互作用，而甲基转移酶 NSUN2 仅识别基序 CAGUGAGC。除了上述发夹之外的 RNA 发夹既不能拉出 YB-1 也不能拉出 NSUN2。这两种蛋白质都存在于 HEK293 细胞分泌的外泌体中。尽管 YB-1 被检测为一种比与上述 RNA 发夹结合的 YB-1 具有稍高电泳迁移率的形式，但假定在蛋白从细胞质转移到外泌体过程中发生了翻译后修饰的变化。总外泌体 RNA（eRNA）的下一代测序揭示了一组大量的 RNA 物种，包括含有上述基序的 mRNA。这类 mRNA 在 exosomes 中的富集程度强烈依赖于 eRNA 特定基序在 mRNA 序列中的位置。总之，我们的研究结果表明 YB-1 和 NSUN2 可能是特定 mRNAs 转移到外泌体的过程的介导者，这使我们推测这些蛋白通过识别上述基序参与了 mRNA 的分选。
