MUTATIONAL ANALYSIS OF THE PRP4 PROTEIN OF SACCHAROMYCES-CEREVISIAE SUGGESTS DOMAIN-STRUCTURE AND SNRNP INTERACTIONS
MUTATIONAL ANALYSIS OF THE PRP4 PROTEIN OF SACCHAROMYCES-CEREVISIAE SUGGESTS DOMAIN-STRUCTURE AND SNRNP INTERACTIONS
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DOI：
10.1093/nar/22.9.1724
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发表时间：
1994-05-11
影响因子：
14.9
通讯作者：
FRIESEN, JD
中科院分区：
文献类型：
--
作者：
HU, J;XU, Y;FRIESEN, JD
酿酒酵母的PRP4蛋白是U4/U6 snRNP的重要组成部分，是mRNA剪接装置的一个组成部分。作为一种确定PRP4蛋白结构-功能关系的方法，我们通过随机和定点诱变分离了50多个PRP4基因的新等位基因，并分析了其中许多等位基因的表型。产生温度敏感型（ts）或零表型的14个单点突变中有12个位于PRP4基因中与蛋白质的β -转导蛋白样区域相对应的部分；其余两个位于基因的中心部分，其中一个位于富含精氨酸-赖氨酸的区域。在氨基端和羧基端分离出9个额外的缺失或缺失/插入突变。这些数据表明，PRP4的氨基端区域（108个氨基酸）是非必需的，而羧基端区域直到倒数第二个氨基酸都是必需的。一个完整的β -转导蛋白样重复序列的缺失（五个重复序列中的第三个）导致表型无效。在非允许温度下，所有ts突变体在体内U3 snRNA初级转录物的剪接上都表现出第一步缺陷。也研究了突变体prp4基因自身拷贝数的增加以及U4/U6 snRNP其他组成部分（PRP3和Us snRNA）的基因拷贝数的增加对prp4突变体生长的影响。我们认为，PRP4蛋白至少有三个结构域：一个由至少108个氨基酸组成的非必需氨基末端，一个由大约140个残基组成的中心基本区域，相对容易发生突变，可能参与RNA相互作用；一个由大约210个残基组成的必需羧基末端区域，具有5个类似于β -转导素的重复区域，可能参与蛋白质-蛋白质相互作用。根据这些结果提出了snRNP组分相互作用的模型。
The PRP4 protein of Saccharomyces cerevisiae is an essential part of the U4/U6 snRNP, a component of the mRNA splicing apparatus. As an approach to the determination of structure - function relationships in the PRP4 protein, we have isolated more than fifty new alleles of the PRP4 gene through random and site-directed mutagenesis, and have analyzed the phenotypes of many of them. Twelve of the fourteen single-point mutations that give rise to temperature-sensitive (ts) or null phenotypes are located in the portion of the PRP4 gene that corresponds to the beta-transducin-like region of the protein; the remaining two are located in the central portion of the gene, one of them in an arginine - lysine-rich region. Nine additional deletion or deletion/insertion mutations were isolated at both the amino- and carboxy-termini. These data show that the amino-terminal region (108 amino acids) of PRP4 is non-essential, while the carboxy-terminal region is essential up to the penultimate amino acid. A deletion of one entire beta-transducin-like repeat (the third of five) resulted in a null phenotype. All ts mutants show a first-step defect in the splicing of U3 snRNA primary transcript in vivo at the non-permissive temperature. The effects on prp4 mutant growth of increased copy-number of mutant prp4 genes themselves, and of genes for other components of the U4/U6 snRNP (PRP3 and Us snRNA) have also been studied. We suggest that the PRP4 protein has at least three domains: a non-essential amino-terminal segment of at least 108 amino acids, a central basic region of about 140 residues that is relatively refractile to mutation and might be involved in RNA interaction, and an essential carboxy-terminal region of about 210 residues with the five repeat-regions that are similar to beta-transducins, which might be involved in protein - protein interaction. A model of interactions of snRNP components suggested by these results is presented.