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

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.

Microtubule integrity regulates budding yeast RAM pathway gene expression

“…However, some ssd1∆ phenotypes, such as zymolyase sensitivity, cellular aggregation, and thermosensitivity, are fully complemented by ssd1-d2 ( Mir et al, 2009 ). In addition, recent structural work demonstrated that Ssd1 binds RNA along the surface of its N-terminus in a region still intact in ssd1-d2 ( Bayne et al, 2022 ). These findings suggest ssd1-d2 is likely at least partially functional.…”

Limitations of composability of cis-regulatory elements in messenger RNA

“…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 (61), which binds the 5’UTRs of their transcripts (11, 62). It is thought that these transcripts are translationally activated at bud sites after the phosphorylation of Ssd1 by a localised kinase, Cbk1 (61).…”

“…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 (62) or motifs that must be repeated in the same transcript to be effective (68, 73). Common motif searching approaches, such as MEME Suite (14), have extended beyond fixed length, gapless motifs but may still assume motifs occur independently and only once per transcript (74).…”

Posttranscriptional regulation of cell wall integrity in budding yeast

“…A previous RNA-immunoprecipitation and microarray screen identified several RNA binding proteins (RBPs), including Bfr1, Hek2, Mrn1, Nab6, Pub1, Scp160, and Ssd1, that preferentially associate with cell wall-related mRNAs (Hogan et al , 2008). The best characterized of these proteins is Ssd1, which represses translation initiation of specific cell wall mRNAs, probably via its interactions with the 5’ UTRs of these transcripts (Bayne et al , 2022; Hu et al , 2018; Jansen et al , 2009). Notably, the ssd1Δ knockout confers sensitivity to compounds targeting the cell wall (Bayne et al , 2022; Jansen et al , 2009; Lee et al , 2014), and synergistically impairs growth when combined with mutations in the CWI pathway (Costanzo et al , 2016).…”

“…The best characterized of these proteins is Ssd1, which represses translation initiation of specific cell wall mRNAs, probably via its interactions with the 5’ UTRs of these transcripts (Bayne et al , 2022; Hu et al , 2018; Jansen et al , 2009). Notably, the ssd1Δ knockout confers sensitivity to compounds targeting the cell wall (Bayne et al , 2022; Jansen et al , 2009; Lee et al , 2014), and synergistically impairs growth when combined with mutations in the CWI pathway (Costanzo et al , 2016). Other RBPs targeting cell wall mRNAs have not yet been investigated in detail, but may have similar roles in regulating cell wall growth and integrity…”