Differential effects of aromatic and charged residue substitutions in the RNA binding domains of the yeast poly(A) binding protein
Differential effects of aromatic and charged residue substitutions in the RNA binding domains of the yeast poly(A) binding protein
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DOI：
10.1006/jmbi.1997.1013
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发表时间：
1997-05-30
影响因子：
5.6
通讯作者：
Sachs, AB
中科院分区：
文献类型：
--
作者：
Deardorff, JA;Sachs, AB
酵母聚(A)结合蛋白（Pab1p）含有4个RNA识别基序（RRMs）。将位点定向突变引入这些RRMs中，以研究它们对特异性和非特异性RNA结合的相对贡献，并确定这些突变对Pab1p支持生存能力的影响。具体来说，在每个RRM中，预测参与RNA结合的带电残基和芳香残基发生了突变。这些突变表明，第二个RRM主要负责poly(A)结合，而第四个RRM主要负责非特异性多聚嘧啶RNA结合。每个RRM中突变的芳香残基对两种结合模式都有贡献，而突变的带电残基主要对非特异性RNA结合有贡献。体内RNA结合与体外结合测量结果相关。此外，RNA结合，而不是高亲和力的聚(A)结合，与Pab1p维持酵母细胞活力的能力相关。这些数据表明，单次芳香族取代可显著降低Pab1p的RNA结合能力，Pab1p结合poly(a)和其他RNA的能力是由不同RRMs内不同残基介导的，并且Pab1p不需要高亲和力的poly(a)尾部结合来发挥其基本功能。(C) 1997学术出版社有限公司
The yeast poly(A)-binding protein (Pab1p) contains four RNA recognition motifs (RRMs). Site-directed mutations were introduced into each of these RRMs in order to investigate their relative contributions to specific and non-specific RNA binding, and to determine the consequences of these mutations on the ability of Pab1p to support viability. Specifically, a charged and an aromatic residue that were predicted to be involved in RNA binding were mutated in each RRM. These mutations revealed that the second RRM is primarily responsible for poly(A) binding, while the fourth RRM is primarily responsible for non-specific polypyrimidine RNA binding. The mutated aromatic residues in each RRM contributed to both modes of binding whereas the mutated charged residues contributed primarily to non-specific RNA binding. RNA binding in vivo correlated with the in vitro binding measurements. Furthermore, RNA binding, but not high-affinity poly(A) binding, correlated with the ability of Pab1p to sustain yeast cell viability. These data suggest that a single aromatic substitution in Pab1p can significantly reduce its RNA binding ability, that the capacity of Pab1p to bind poly(A) as well as other RNAs is mediated by distinct residues within different RRMs, and that Pab1p does not require high affinity poly(A) tail binding to perform its essential function. (C) 1997 Academic Press Limited.