How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing
How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing
复制标题
DOI：
10.1017/s1355838202022033
复制
发表时间：
2002-08-01
期刊：
影响因子：
4.5
通讯作者：
Schwer, B
中科院分区：
文献类型：
--
作者：
James, SA;Turner, W;Schwer, B
SIu 7和Prp 18在酵母前体mRNA剪接的第二步中协同作用。在这里，我们发现382个氨基酸的SIu 7蛋白包含两个功能重要的结构域：锌关节（（122）CRNCGEAGHKEKDC（135））和Prp 18相互作用结构域（（EIELMKLELY 224）-E-215）。丙氨酸簇突变（EIE 217）-E-215和（LELY 224）-L-221废除SIu 7结合Prp 18在双杂交试验和体外，并引发温度敏感的生长表型在体内。然而，突变对SIu 7在体外前体mRNA剪接中的功能没有影响。锌关节残基Cys 122，His 130和Cys 135的单个丙氨酸突变对细胞生长没有影响，但在体外前体mRNA剪接过程中导致SIu 7功能依赖于Prp 18。具体而言，锌关节突变体需要Prp 18，以结合剪接体。两个SIu 7结构域中的复合突变（例如，C122 A-EIE、H130 A-EIE和C135 A-EIE）在体内是致命的，在体外消除剪接，表明SIu 7和Prp 18之间的物理相互作用对于剪接中的合作是重要的。与免疫沉淀结合的消耗/重建研究表明，第二步因子以以下顺序被募集到剪接体：SIu 7-> Prp 18-> Prp 22。所有三种蛋白质在第2步后从剪接体释放，伴随着成熟mRNA的释放。
SIu7 and Prp18 act in concert during the second step of yeast pre-mRNA splicing. Here we show that the 382-amino-acid SIu7 protein contains two functionally important domains: a zinc knuckle ((122)CRNCGEAGHKEKDC(135)) and a Prp18-interaction domain ((EIELMKLELY224)-E-215). Alanine cluster mutations of (EIE217)-E-215 and (LELY224)-L-221 abrogated SIu7 binding to Prp18 in a two-hybrid assay and in vitro, and elicited temperature-sensitive growth phenotypes in vivo. Yet, the mutations had no impact on SIu7 function in pre-mRNA splicing in vitro. Single alanine mutations of zinc knuckle residues Cys122, His130, and Cys135 had no effect on cell growth, but caused SIu7 function during pre-mRNA splicing in vitro to become dependent on Prp18. Specifically, zinc knuckle mutants required Prp18 in order to bind to the spliceosome. Compound mutations in both SIu7 domains (e.g., C122A-EIE, H130A-EIE, and C135A-EIE) were lethal in vivo and abolished splicing in vitro, suggesting that the physical interaction between SIu7 and Prp18 is important for cooperation in splicing. Depletion/reconstitution studies coupled with immunoprecipitations suggest that second step factors are recruited to the spliceosome in the following order: SIu7 --> Prp18 --> Prp22. All three proteins are released from the spliceosome after step 2 concomitant with release of mature mRNA.