ABSTRACT

During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

摘要

在剪接体组装过程中，使内含子的 5' 和 3' 末端靠近的相互作用对于成熟 mRNA 的产生至关重要。在这里，我们报告了人类 U1 小核 RNA (snRNA) 的茎环 3 (SL3) 和 4 (SL4) 在维持最佳 U1 snRNP 功能以及与 U2 snRNP 形成交叉内含子接触方面的协同作用。我们发现 SL3 和 SL4 结合不同的剪接体蛋白并将 U1 snRNA 活性测定与 siRNA 介导的敲低相结合，我们证明 SL3 和 SL4 分别通过 RNA 解旋酶 UAP56 和 U2 蛋白 SF3A1 起作用。体外使用 UV 交联和剪接分析的分析表明，SL3 可能促进 SL4-SF3A1 相互作用，从而增强 A 复合物的形成和前 mRNA 剪接。总体而言，这些结果突出了 SL3 和 SL4 的不同接触在人类剪接体组装的早期步骤中在桥接前 mRNA 结合的 U1 和 U2 snRNP 中的重要作用。