The conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not well understood. Here we present the crystal structure of Ssd1 at 1.9 Å resolution. Active RNase II enzymes have a characteristic, internal RNA binding path, but in Ssd1 this is blocked by remnants of regulatory sequences. Instead, RNA binding activity has likely been relocated to the outer surface of the protein. Using in vivo crosslinking and cDNA analysis (CRAC), we identify Ssd1-RNA binding sites. These are strongly enriched in 5′UTRs of a subset of mRNAs encoding cell wall proteins. Based on these and previous analyses, we identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory elements in the RNase II family.

中文翻译：

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酵母Ssd1是RNase II家族的非酶成员，具有替代的RNA识别接口

保守的真菌RNA结合蛋白Ssd1在应激反应，细胞分裂和毒力中很重要。Ssd1与核酸酶RNase II家族的Dis3L2密切相关，但缺乏催化活性，可能通过抑制相关mRNA的翻译起作用。先前的研究确定了富含Ssd1相关转录物的基序，但对Ssd1结合的序列要求还不甚了解。在这里，我们介绍了1.9Å分辨率的Ssd1的晶体结构。活性RNase II酶具有特征性的内部RNA结合途径，但在Ssd1中，这被调控序列的残余物所阻断。相反，RNA结合活性可能已重新定位到蛋白质的外表面。使用体内交联和cDNA分析（CRAC），我们确定Ssd1-RNA结合位点。这些在编码细胞壁蛋白的mRNA子集中的5'UTR中高度富集。基于这些和以前的分析，我们确定了一个保守的二聚体基序，该基序在体外以高亲和力结合Ssd1。这些研究为理解基因表达的多效转录后调节因子的功能提供了新的框架，并为RNase II家族调节因子的进化提供了见识。