Frilander Mikko J, Meng Xiaojuan
Institute of Biotechnology, Program on Developmental Biology, PL56 (Viikinkaari 9), 00014 University of Helsinki, Finland.
Mol Cell Biol. 2005 Jun;25(12):4813-25. doi: 10.1128/MCB.25.12.4813-4825.2005.
U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5' end of the U12 snRNA and the -2 position upstream of the 5' splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5' exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5' exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5' splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5' exon sequences near 5' splice site and the branch point sequence, suggesting that the 5' splice site and branch point sequences are separated by <40 to 50 A in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.
U12 snRNA是U12依赖型剪接体中分支点识别所必需的。利用位点特异性交联，我们捕捉到了U12 snRNA的5'端与P120和SCN4a剪接底物5'剪接位点上游-2位置之间意外的相互作用。与5'外显子接触的U12 snRNA核苷酸与在剪接体催化核心中与U6atac snRNA形成催化重要的螺旋Ib的核苷酸相同。然而，U12/5'外显子相互作用是短暂的，发生在U4atac/U6atac.U5三小核核糖核蛋白进入剪接体之前。这表明U12 snRNA的螺旋Ib区域在剪接体组装早期就定位在5'剪接位点附近，只是后来才与U6atac相互作用形成螺旋Ib。我们还提供了证据表明，U12 snRNA可以同时与5'剪接位点附近的5'外显子序列和分支点序列相互作用，这表明在U12依赖型剪接体的A复合物中，5'剪接位点和分支点序列之间的距离小于40至50埃。因此，在催化的第一步中，随后不需要进行重大重排来定位这些位点。
