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.

先前我们已经证明YB-1是HEK293细胞胞质（S100）提取物中唯一与RNA发夹特异性相互作用的蛋白，每个发夹均包含ACCAGCCU（1），CAGUGAGC（2）和UAAUCCCA（3）其中一个基序被鉴定为通常在外泌体RNA中发现，并被提议为潜在的将RNA靶向外泌体的顺式作用元件。在这里，我们探讨了YB-1与septin 14 mRNA（SEPT14 RNA）的3'-非翻译区（UTR）片段的相互作用，该片段包含所有三个基序。我们证明了使用生物素化的SEPT14 RNA从HEK293 S100提取物中提取的蛋白质中存在YB-1。使用重组YB-1，发现SEPT14 RNA可以以协作方式结合每摩尔RNA最多5摩尔蛋白质，这被证明主要是由于上述基序的存在。根据YB-1对这些RNA发夹的亲和力，带有基序1和2的RNA发夹与SEPT14 RNA竞争与蛋白质的结合，而带有基序3的RNA发夹的竞争则较弱。使用YB-1结合的RNA，在所有三个基序中都揭示了免受羟基自由基攻击的核苷酸，尽管带有基序2的发夹（尤其是具有基序1的发夹）在基序外包含许多受保护的核苷酸，这表明这些基序的特定环境显着YB-1绑定。对从RNA-seq数据获得的HEK293细胞mRNA 3'UTR中的1-3模体环境的分析导致我们得出结论，即3'UTR中YB-1的主要结合位点是包含某些基序的发夹以及其特定的环境；得出了这些发夹的共有序列。因此，