Puig O, Gottschalk A, Fabrizio P, Séraphin B
Gene Expression Program, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.
Genes Dev. 1999 Mar 1;13(5):569-80. doi: 10.1101/gad.13.5.569.
Intron definition and splice site selection occur at an early stage during assembly of the spliceosome, the complex mediating pre-mRNA splicing. Association of U1 snRNP with the pre-mRNA is required for these early steps. We report here that the yeast U1 snRNP-specific protein Nam8p is a component of the commitment complexes, the first stable complexes assembled on pre-mRNA. In vitro and in vivo, Nam8p becomes indispensable for efficient 5' splice site recognition when this process is impaired as a result of the presence of noncanonical 5' splice sites or the absence of a cap structure. Nam8p stabilizes commitment complexes in the latter conditions. Consistent with this, Nam8p interacts with the pre-mRNA downstream of the 5' splice site, in a region of nonconserved sequence. Substitutions in this region affect splicing efficiency and alternative splice site choice in a Nam8p-dependent manner. Therefore, Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence. This supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.
内含子定义和剪接位点选择发生在剪接体组装的早期阶段，剪接体是介导前体mRNA剪接的复合体。U1 snRNP与前体mRNA的结合是这些早期步骤所必需的。我们在此报告，酵母U1 snRNP特异性蛋白Nam8p是起始复合体的一个组成部分，起始复合体是在mRNA前体上组装的首个稳定复合体。在体外和体内，当由于存在非典型5'剪接位点或缺乏帽结构而导致这一过程受损时，Nam8p对于高效识别5'剪接位点变得不可或缺。在后者情况下，Nam8p可稳定起始复合体。与此一致的是，Nam8p在5'剪接位点下游的非保守序列区域与前体mRNA相互作用。该区域的替换以依赖Nam8p的方式影响剪接效率和可变剪接位点的选择。因此，Nam8p参与了一种新机制，通过该机制，一个snRNP组分可通过与非保守序列相互作用来影响剪接位点选择并调节内含子去除。这支持了一个模型，即早期5'剪接识别源于剪接机制与前体mRNA不同区域建立的相互作用网络。
