Sharma Shalini, Wongpalee Somsakul Pop, Vashisht Ajay, Wohlschlegel James A, Black Douglas L
Department of Basic Medical Sciences, University of Arizona, College of Medicine-Phoenix, Phoenix, Arizona 85004, USA; Department of Microbiology, Immunology, and Molecular Genetics.
Molecular Biology Interdepartmental Graduate Program.
Genes Dev. 2014 Nov 15;28(22):2518-31. doi: 10.1101/gad.248625.114.
The pairing of 5' and 3' splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 5' splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3' splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5' and 3' splice site complexes within the assembling spliceosome.
内含子两端5'和3'剪接位点的配对是剪接体形成及其调控过程中的关键步骤。在剪接体组装过程中，将两个剪接位点聚集在一起的相互作用必须高度特异且准确，以确保功能性mRNA的表达并做出特定的可变剪接选择。在此，我们报道了识别5'剪接位点的U1小核RNA（snRNA）的茎环4（SL4）与U2小核糖核蛋白颗粒（snRNP）复合体的一个组分之间的新相互作用，该复合体在3'剪接位点处跨内含子组装。通过U1 snRNP互补分析，我们发现SL4在体内剪接过程中必不可少。在体外剪接反应中添加游离的U1-SL4会抑制剪接，并在形成前剪接体A复合体之前阻止复合体组装，这表明在剪接体组装过程中需要SL4的接触。为了表征这种RNA结构的相互作用，我们结合使用了细胞培养中氨基酸稳定同位素标记（SILAC）、生物素/中性抗生物素蛋白亲和下拉和质谱分析。我们表明U1-SL4与U2 snRNP的SF3A1蛋白相互作用。我们发现U1 snRNA与SF3A1之间的这种相互作用发生在组装于前体mRNA上的前剪接体复合体内。因此，U1 snRNA的SL4对剪接很重要，其与SF3A1的相互作用介导了组装中的剪接体内5'和3'剪接位点复合体之间的接触。
