The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex
The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex
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DOI：
10.1017/s135583829999012x
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发表时间：
1999-07-01
期刊：
影响因子：
4.5
通讯作者：
Rossi, JJ
中科院分区：
文献类型：
--
作者：
Kim, DH;Rossi, JJ
酵母PRP44基因，或命名为Brr2，SLT22，RSS1，或SNU246，在前mRNA剪接过程中编码一个246 kDa的蛋白质，推测具有RNA解旋酶功能。该蛋白是典型的DEAD/H家族成员，但与该家族的大多数其他成员不同的是，它含有两个假定的RNA解旋酶结构域，每个结构域都有一个高度保守的ATPase基序。在这项研究之前，人们对这两个领域的功能角色知之甚少。我们提供了遗传和生化证据，证明只有第一个解旋酶结构域的ATPase基序是细胞存活和前mRNA剪接所必需的。第一个结构域的突变过表达会导致显性的阴性表型，这些突变株的提取物在体外抑制前mRNA的剪接。亲和纯化蛋白的体外分析表明，只有第一个解旋酶结构域具有聚(U)依赖的ATPase活性。在体内过表达显性负蛋白会降低游离U4和U6 SnRNA的相对丰度，并伴随着U4/U6双链的积累。野生型Prp44p的过表达缓解了U4/U6双链的积累。三种DEAD/H盒蛋白Prp16p、Prp22p和Prp44p已被证明在体外影响U4/U6解离活性。在这些细胞中诱导表达ATPase结构域突变后，我们探索了这些蛋白在体内介导这一反应的可能作用。虽然Prp16p、Prp22p或Prp44p突变形式的过度表达是致命的，但只有突变Prp44p的表达导致U4/U6螺旋的积累。我们的结果，当结合以前发表的体外结果，支持Prp44p在解开U4/U6螺旋中的直接作用。
The yeast PRP44 gene, alternatively named as BRR2, SLT22, RSS1, or SNU246, encodes a 246-kDa protein with putative RNA helicase function during pre-mRNA splicing. The protein is a typical DEAD/H family member, but unlike most other members of this family, it contains two putative RNA helicase domains, each with a highly conserved ATPase motif. Prior to this study little was known about functional roles for these two domains. We present genetic and biochemical evidence that ATPase motifs of only the first helicase domain are required for cell viability and pre-mRNA splicing. Overexpression of mutations in the first domain results in a dominant negative phenotype, and extracts from these mutant strains inhibit in vitro pre-mRNA splicing. In vitro analyses of affinity purified proteins revealed that only the first helicase domain possesses poly (U)-dependent ATPase activity. Overexpression of a dominant negative protein in vivo reduces the relative abundance of free U4 and U6 snRNA with a concomitant accumulation of the U4/U6 duplex. Accumulation of the U4/U6 duplex was relieved by overexpression of wild-type Prp44p. Three DEAD/H box proteins, Prp16p, Prp22p and Prp44p, have previously been shown to affect U4/U6 unwinding activity in vitro. The possible role of these proteins in mediating this reaction in vivo was explored following induced expression of ATPase domain mutants in each of these. Although overexpression of the mutant form of either Prp16p, Prp22p, or Prp44p was lethal, only expression of the mutant Prp44p resulted in accumulation of the U4/U6 helix. Our results, when combined with previously published in vitro results, support a direct role for Prp44p in unwinding of the U4/U6 helix.