Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

Abstract

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.

Repeated Evolution of Inactive Pseudonucleases in a Fungal Branch of the Dis3/RNase II Family of Nucleases

“…For example, access to the active site may be blocked by mutations that occlude the RNA-binding channel. Indeed, our structure of Sc Ssd1 indicates that access to the former active site is blocked by loop insertions in multiple domains ( Bayne et al. 2020 ).…”

“…2009 ). We recently solved the structure of Sc Ssd1, the fold of which is conserved with Dis3 and Dis3L2 ( Bayne et al. 2020 ).…”

Interplay of the RNA Exosome Complex and RNA-Binding Protein Ssd1 in Maintaining Cell Wall Stability in Yeast

“…Inactivation of both proteins would then have a sequential, rather than a parallel, effect on cell wall instability at high temperatures. To this end, we examined relevant transcript levels in the RNA sequencing data set of BY4741 rrp6 Δ mutant cells (10 min at 42°C) ( 23 ) and the enrichment of Ssd1 binding to mRNAs from the CRAC (cross-linking and analysis of cDNAs) data set (16 min at 42°C) ( 24 ). The first question we asked was how many cell-wall-related transcripts are significantly enriched for Ssd1 binding at high temperature?…”

Limitations of composability of cis-regulatory elements in messenger RNA

“…Reductionist search algorithms that attempt to discover the shortest possible sequence contributing to expression may limit the discovery of novel CREs. High resolution maps of protein-RNA interactions are revealing RBPs with gapped, multi-partite motifs (46) or motifs that must be repeated in the same transcript to be effective (52,57). Common motif searching approaches, such as MEME Suite ( 13), have extended beyond fixed length, gapless motifs but may still assume motifs occur independently and only once per transcript (58).…”

“…Another example where the effect of a CRE on a transcript depends on co-localisation with a regulatory kinase comes from the fungal RNA-binding protein Ssd1. Yeast cell wall proteins such as Sun4 and Tos6 are translationally repressed by Ssd1 ( 45), which binds the 5'UTRs of their transcripts (10,46). It is thought that these transcripts are translationally activated at bud sites after the phosphorylation of Ssd1 by a localised kinase, Cbk1 (45).…”