Deep mutational scanning of an RRM domain of the Saccharomyces cerevisiae poly(A)-binding protein.
Deep mutational scanning of an RRM domain of the Saccharomyces cerevisiae poly(A)-binding protein.
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DOI：
10.1261/rna.040709.113
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发表时间：
2013-11
期刊：
影响因子：
--
通讯作者：
Fields S
中科院分区：
文献类型：
--
作者：
Melamed D;Young DL;Gamble CE;Miller CR;Fields S
RNA识别基序(RRM)是真核生物中最常见的RNA结合域，其氨基酸序列的不同影响着结合的特异性和亲和力。这项研究应用了一种深度突变扫描方法来对酵母聚(A)结合蛋白Pab1的RRM2结构域的100,000多个变体的功能活性进行评分。作者使用这些数据来完善目前对Pab1结构域和RRM结构域的理解。RNA识别基序(RRM)是真核生物中最常见的RNA结合域。RRM序列的差异在一定程度上决定了RNA和蛋白质结合的特异性和亲和力。我们用深度突变扫描的方法研究了酿酒酵母聚(A)结合蛋白(Pab1)的RRM2结构域的序列-功能关系。通过对100,000多个独特的Pab1变体的活性进行评分，包括1246个单一氨基酸替换，我们描绘了每个残基的突变限制。具有相似突变模式的残基的聚集显示了三大类，主要由RNA结合残基、疏水核心残基和剩余残基组成。第一类还包括一个高度保守的残基，G150，它不参与RNA结合，它可以突变来破坏Pab1的稳定。酵母Pab1残基对其进化保守性的突变敏感性的比较表明，大多数残基比自然序列中存在的耐受更多的取代，尽管其他耐受较少取代的残基可能指向酵母中的特殊功能。对∼40,000双突变体的分析表明，两个表现出上位相互作用的突变之间的距离较短。作为相互作用的例子，N139T、N139S和I157L突变抑制了其他干扰RNA结合和蛋白质稳定性的突变。总体而言，这项研究表明，活细胞可以接受一次测试，并行分析数十万种蛋白质变体。
The RNA recognition motif (RRM) is the most common eukaryotic RNA-binding domain, with differences in its amino acid sequence affecting binding specificity and affinity. This study applied a deep mutational scanning approach to score the functional activity of more than 100,000 variants of the RRM2 domain of the yeast poly(A)-binding protein, Pab1. The authors use these data to refine the current understanding of the Pab1 domain and of RRM domains in general. The RNA recognition motif (RRM) is the most common RNA-binding domain in eukaryotes. Differences in RRM sequences dictate, in part, both RNA and protein-binding specificities and affinities. We used a deep mutational scanning approach to study the sequence-function relationship of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein (Pab1). By scoring the activity of more than 100,000 unique Pab1 variants, including 1246 with single amino acid substitutions, we delineated the mutational constraints on each residue. Clustering of residues with similar mutational patterns reveals three major classes, composed principally of RNA-binding residues, of hydrophobic core residues, and of the remaining residues. The first class also includes a highly conserved residue not involved in RNA binding, G150, which can be mutated to destabilize Pab1. A comparison of the mutational sensitivity of yeast Pab1 residues to their evolutionary conservation reveals that most residues tolerate more substitutions than are present in the natural sequences, although other residues that tolerate fewer substitutions may point to specialized functions in yeast. An analysis of ∼40,000 double mutants indicates a preference for a short distance between two mutations that display an epistatic interaction. As examples of interactions, the mutations N139T, N139S, and I157L suppress other mutations that interfere with RNA binding and protein stability. Overall, this study demonstrates that living cells can be subjected to a single assay to analyze hundreds of thousands of protein variants in parallel.
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影响因子：
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作者：
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DOI：
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