Kyburz Andrea, Friedlein Arno, Langen Hanno, Keller Walter
Department of Cell Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Mol Cell. 2006 Jul 21;23(2):195-205. doi: 10.1016/j.molcel.2006.05.037.
Eukaryotic pre-mRNAs are capped at their 5' ends, polyadenylated at their 3' ends, and spliced before being exported from the nucleus to the cytoplasm. Although the three processing reactions can be studied separately in vitro, they are coupled in vivo. We identified subunits of the U2 snRNP in highly purified CPSF and showed that the two complexes physically interact. We therefore tested whether this interaction contributes to the coupling of 3' end processing and splicing. We found that CPSF is necessary for efficient splicing activity in coupled assays and that mutations in the pre-mRNA binding site of the U2 snRNP resulted in impaired splicing and in much reduced cleavage efficiency. Moreover, we showed that efficient cleavage required the presence of the U2 snRNA in coupled assays. We therefore propose that the interaction between CPSF and the U2 snRNP contributes to the coupling of splicing and 3' end formation.
真核生物前体mRNA在其5'端进行加帽，在其3'端进行多聚腺苷酸化，并在从细胞核输出到细胞质之前进行剪接。尽管这三种加工反应可以在体外分别进行研究，但它们在体内是偶联的。我们在高度纯化的CPSF中鉴定出U2 snRNP的亚基，并表明这两种复合物存在物理相互作用。因此，我们测试了这种相互作用是否有助于3'端加工和剪接的偶联。我们发现在偶联实验中，CPSF对于高效剪接活性是必需的，并且U2 snRNP的前体mRNA结合位点发生突变会导致剪接受损以及切割效率大幅降低。此外，我们表明在偶联实验中高效切割需要U2 snRNA的存在。因此，我们提出CPSF与U2 snRNP之间的相互作用有助于剪接和3'端形成的偶联。
