U4/U6.U5三小核核糖核蛋白复合体中65 kDa和110 kDa的富含丝氨酸/精氨酸的蛋白对于成熟剪接体的组装至关重要。
The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes.
作者信息
Makarova O V, Makarov E M, Lührmann R
机构信息
Max-Planck-Institute for Biophysical Chemistry, Department of Cellular Biochemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
出版信息
EMBO J. 2001 May 15;20(10):2553-63. doi: 10.1093/emboj/20.10.2553.
The association of the U4/U6.U5 tri-snRNP with pre-spliceosomes is a poorly understood step in the spliceosome assembly pathway. We have identified two human tri-snRNP proteins (of 65 and 110 kDa) that play an essential role in this process. Characterization by cDNA cloning of the 65 and 110 kDa proteins revealed that they are likely orthologues of the yeast spliceosomal proteins Sad1p and Snu66p, respectively. Immunodepletion of either protein from the HeLa cell nuclear extracts inhibited pre-mRNA splicing due to a block in the formation of mature spliceosomes, but had no effect on the integrity of the U4/U6.U5 tri-snRNP. Spliceosome assembly and splicing catalysis could be restored to the respective depleted extract by the addition of recombinant 65 or 110 kDa protein. Our data demonstrate that both proteins are essential for the recruitment of the tri-snRNP to the pre-spliceosome but not for the maintenance of the tri-snRNP stability. Moreover, since both proteins contain an N-terminal RS domain, they could mediate the association of the tri-snRNP with pre-spliceosomes by interaction with members of the SR protein family.
U4/U6.U5三小核核糖核蛋白与前剪接体的结合是剪接体组装途径中一个尚未被充分理解的步骤。我们鉴定出了两种人类三小核核糖核蛋白（分别为65 kDa和110 kDa），它们在这一过程中发挥着至关重要的作用。通过对65 kDa和110 kDa蛋白进行cDNA克隆分析发现，它们可能分别是酵母剪接体蛋白Sad1p和Snu66p的直系同源物。从HeLa细胞核提取物中免疫去除任何一种蛋白都会抑制前体mRNA剪接，原因是成熟剪接体的形成受阻，但对U4/U6.U5三小核核糖核蛋白的完整性没有影响。通过添加重组的65 kDa或110 kDa蛋白，可以使剪接体组装和剪接催化恢复到各自的缺失提取物中。我们的数据表明，这两种蛋白对于将三小核核糖核蛋白招募到前剪接体中至关重要，但对维持三小核核糖核蛋白的稳定性并非必需。此外，由于这两种蛋白都含有一个N端RS结构域，它们可能通过与SR蛋白家族成员相互作用来介导三小核核糖核蛋白与前剪接体的结合。
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