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

Abstract

The RNase II family of 3′–5′ exoribonucleases is present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like “pseudonucleases.” The latter function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. However, the evolutionary origins of these pseudonucleases are unknown: What sequence of events led to their novel function, and when did these events occur? Here, we show how RNase II pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. In contrast, N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the nonnuclease function requires these regions. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid “titan” growth stages. This phenotype of C. neoformans Ssd1/Dis3L2 deletion is consistent with those of inactive fungal pseudonucleases, yet the protein retains an active site sequence signature. We propose that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, whereas the RNase activity was lost repeatedly in independent lineages.

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

“…The binding motif of Ssd1 is also conserved at longer evolutionary distances. Indeed, a sequence similar to the SEE motif was reported to be found in 5′UTRs of S. pombe Sts5, a homologue of Ssd1 that is also a pseudonuclease ( 4 , 71 ). We focused our search on homologues of SUN4; SUN4 and SIM1 are post-whole genome duplication paralogues, as are UTH1 and NCA3 ( 72 ).…”

“…We demonstrate that Ssd1p recognizes a bipartite element that encompasses the previously identified SEE motif, along with a second upstream motif. We present a 1.9 Å X-ray crystal structure of S. cerevisiae Ssd1p and show that it retains the domain architecture of RNase II proteins ( 1 , 4 , 26 ). The absence of enzymatic activity in Ssd1p is revealed to arise from both mutation of active site residues and the fixing in place of loop elements that likely regulate activity of other DIS3 family enzymes.…”

“…However, some RNase II family proteins are pseudonucleases with regulatory roles in RNA metabolism, rather than active enzymes. These include the fungal Ssd1 family that is closely related to Dis3L2 ( 4 ). Saccharomyces cerevisiae Ssd1 binds RNA, but does not have detectable exonuclease activity ( 5 ).…”

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

“…One possible candidate was the polymorphic SSD1 gene, which encodes the RNA-binding protein Ssd1, which binds 5′ and 3′ UTRs of mRNAs encoding cell wall morphogenesis proteins and represses their translation ( 5 , 6 ). The Ssd1 family is closely related to the Dis3L2 3′→5′ exonucleases, which belong to the same RNase II/RNB family as the catalytic subunit of the RNA exosome Dis3 ( 22 ). In the genetic background BY4741, the active SSD1-V allele encodes the full-length protein, whereas strains of genetic background W303 carry the ssd1-d allele, which contains a premature stop codon due to a C-to-G transversion at nucleotide 2094, resulting in termination of the Ssd1 protein at the beginning of its RNB domain ( Fig.…”

Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation

“…Pleiotropic elp3 phenotypes [38,47] are enhanced by the ssd1d allele present in common laboratory strains derived from W303 yeast [30]. SSD1 is evolutionary conserved in the fungal kingdom and its orthologue in the distantly related fission yeast S. pombe similarly encodes a P-body associated RNA-binding protein [48,49]. Thus, the functional conservation of Ssd1 in the fungal kingdom is likely, but phenotypic variation of tRNA modification defects in other fungi remain to be studied.…”