Ansari A, Schwer B
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
EMBO J. 1995 Aug 15;14(16):4001-9. doi: 10.1002/j.1460-2075.1995.tb00071.x.
Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.
了解前体mRNA剪接机制需要对所有相关成分进行表征。在本研究中，我们以遗传和生化定义的酵母PRP16蛋白为出发点，用于鉴定体外第二步催化步骤所需的其他因子。我们通过甘油梯度沉降从缺乏PRP16的酵母提取物中分离出形成的剪接体。该过程将含有套索中间体和外显子1的剪接体复合物与全细胞酵母提取物中存在的游离蛋白质分离。然后，我们用纯化的蛋白质或酵母提取物组分补充这些剪接体，作为第二步剪接因子的功能测定。我们表明，SLU7蛋白和一种我们命名为SSF1（第二步因子1）的新活性与PRP16协同作用，以促进剪接第二步催化步骤的进行。利用PRP16和SLU7功能对ATP的不同需求，我们表明SLU7可以在剪接途径中PRP16之后起作用。
