Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

对前 mRNA 剪接机制的理解受到体内剪接体检查的技术挑战的限制。在这里，我们报告了从哺乳动物细胞核染色质沉淀中溶解的预催化 A 或 B 样剪接体衍生的 RNP 复合物的分离。我们发现这些复合物含有 U2 snRNP 蛋白和一部分与受保护的 RNA 片段结合的 U2 snRNA，这些片段精确地映射到转录组中的内含子分支位点。这些 U2 复合物还包含剪接调节因子 RBM5 和 RBM10。我们发现 RBM5 和 RBM10 与几乎所有分支位点复合体结合，而不仅仅是那些受调控外显子的分支位点复合体。保守的 RBM5/RBM10 肽序列（包括锌指基序）的删除破坏了 U2 相互作用，并使蛋白质失去了抑制许多替代外显子的活性。我们提出了一个模型，其中 RBM5 和 RBM10 作为 U2 snRNP 复合体的组成部分在分支位点碱基配对后调节剪接。