Gottschalk A, Bartels C, Neubauer G, Lührmann R, Fabrizio P
Department of Cellular Biochemistry, Max-Planck-Institute of Biophysical Chemistry, D-37077 Göttingen, Germany.
Mol Cell Biol. 2001 May;21(9):3037-46. doi: 10.1128/MCB.21.9.3037-3046.2001.
We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.
我们已经分离并对Snu17p进行了微测序，它是一种预测分子量为17 kDa的新型酵母蛋白，含有一个RNA识别基序。我们证明Snu17p特异性结合U2小核核糖核蛋白（snRNP），并且它是剪接体的一部分，因为前体mRNA和套索状外显子2能与Snu17p特异性共沉淀。虽然SNU17基因并非必需基因，但其敲除会导致体内生长缓慢的表型以及前体mRNA剪接缺陷。此外，从snu17缺失（snu17Δ）突变体中制备的提取物中，剪接的第一步显著减少。通过添加重组Snu17p可有效逆转此缺陷。为了研究Snu17p发挥作用的剪接体组装步骤，我们使用了非变性凝胶电泳。在缺乏Snu17p的提取物中，剪接体以单一缓慢迁移的复合物形式存在。在野生型提取物中，通常至少会观察到两种不同的复合物：预剪接体或B复合物，包含U2但不包含U1 snRNP；以及具有催化活性的剪接体或A复合物，包含U2、U6和U5 snRNPs。对snu17Δ剪接体的Northern印迹分析和亲和纯化表明，它包含U1、U2、U6、U5和U4 snRNPs。U1 snRNP的意外稳定以及U4 snRNP的未解离表明，Snu17p的缺失抑制了在U1 snRNP释放之前以及添加[U4/U6.U5]三snRNP之后的剪接体组装进程。
