Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

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.

在剪接体组装过程中，将内含子的5'和3'端拉近以促进成熟mRNA产生的相互作用至关重要。本研究报告了人类U1小核RNA（snRNA）的茎环3（SL3）和茎环4（SL4）在维持U1 snRNP最佳功能以及与U2 snRNP形成跨内含子接触中的协同作用。研究发现，SL3和SL4分别与不同的剪接体蛋白结合，并通过将U1 snRNA活性检测与siRNA介导的敲低相结合，我们证实SL3和SL4通过RNA解旋酶UAP56和U2蛋白SF3A1分别发挥作用。利用紫外交联和剪接分析进行的体外研究指出，SL3可能通过促进SL4-SF3A1相互作用，进而增强A复合物形成和前体mRNA的剪接。总的来说，这些结果凸显了SL3和SL4独特的接触点在连接早期人类剪接体组装过程中结合的前体mRNA的U1和U2 snRNPs中的关键作用。