Abstract
Ssd1, a conserved fungal RNA-binding protein, 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 likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5′ untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. 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 and binding elements in the RNase II family.
Highlights
Mechanisms of post-transcriptional control of gene expression by RNA-binding proteins (RBPs) include modulation of mRNA translation and decay
CFW binds to chitin in fungal cell walls, and ssd1Δ strains are sensitive to CFW concentrations in the range of 10–100 M [67]
We find CCAACU motifs upstream of CNYUCNYU motifs in 5 UTRs only from S. cerevisiae and C. albicans homologues, suggesting that the CNYUCNYU part is more broadly conserved and that the bipartite RNA binding motif is particular to the budding yeast clade
Summary
Mechanisms of post-transcriptional control of gene expression by RNA-binding proteins (RBPs) include modulation of mRNA translation and decay. Eukaryotic DIS3 (Rrp44) and Dis3L2 are RNase II family 3 –5 exonucleases. DIS3 or Rrp (for human and yeast orthologues, respectively) is the essential nuclease associated with the eukaryotic exosome complex that processes and/or turns over the majority of cellular RNAs [2]. Dis3L2 is a related nuclease that is specific for RNA substrates with an oligouridine 3 tail [3]. Some RNase II family proteins are pseudonucleases with regulatory roles in RNA metabolism, rather than active enzymes. These include the fungal Ssd family that is closely related to Dis3L2 [4]. Saccharomyces cerevisiae Ssd binds RNA, but does not have detectable exonuclease activity [5]
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