Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA–protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)⁺ RNA–protein crosslinking data provide information on the organization of RNA–protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)⁺ RNA, we combine poly(A)⁺ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3′ end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well.

中文翻译：

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对裂变酵母 poly(A)+ RNA 相互作用组的全系统分析揭示了对 RNA 蛋白复合物的组织和功能的洞察。

大型 RNA 结合复合物在基因表达和协调 mRNA 的产生、功能和周转中发挥核心作用。这些分子机器内 RNA-蛋白质相互作用的准确性和动力学对其功能至关重要，并由 RNA 结合蛋白 (RBP) 介导。在这里，我们展示了裂变酵母全细胞 poly(A) ⁺ RNA-蛋白质交联数据提供了有关 RNA-蛋白质复合物组织的信息。为了评估细胞 RBP 在 poly(A) ⁺ RNA 上的相对富集，我们将 poly(A) ⁺使用全细胞提取标准化程序捕获 RNA 相互作用组。这种方法产生了体内 RNA 结合活性的估计，这些活性可以识别直接接触 RNA 的多蛋白复合物中的亚基。作为验证，我们追踪了 3' 端加工机制的不同功能模块的 RNA 相互作用，并揭示了额外的联系。将我们的分析扩展到 RNA 外泌体复合体的不同突变体，我们探索了通过复合体的底物通道如何受到突变的影响。我们的数据突出了 RNA 解旋酶 Mtl1 在复合物调控中的核心作用，并提供了对不同成分如何促进复合物与底物 RNA 结合的见解。此外，我们描述了在多个物种的 RNA 相互作用组中反复检测到的新型 RBP 的 RNA 结合活性。我们发现其中许多，包括亲环蛋白和硫氧还蛋白，是体内亚化学计量的 RNA 相互作用物。因为 RBPomes 在物种之间表现出非常好的整体一致性，我们建议我们在裂变酵母中观察到的 RNA 结合特征也可能适用于高等真核生物中的相关蛋白质。