Hermann-Le Denmat, S. | bioRxiv Science

bioRxiv2020

The eukaryotic ribosomal protein S15/uS19 is involved in fungal development and its C-terminal tail contributes to stop codon recognition

S15/uS19 is one of the fifteen universally conserved ribosomal proteins of the small ribosomal subunit. While prokaryotic uS19 is located away from the mRNA decoding site, cross-linking studies identified eukaryotic uS19 C-terminal tail as contacting the A site on the 80S ribosome. Here, we study the effects of uS19 mutations isolated as translation accuracy mutations in the filamentous fungus Podospora anserina. All mutations alter residues of uS19 C-terminal tail, and cluster to the eukaryote-specific decapeptide 138-PGIGATHSSR-147. All mutations modify fungal development and cytosolic translation, albeit differently. Two mutations (P138S and S145F) increase fungus longevity and display mild effects on translation, while others (G139D and G139C) decrease longevity, have stronger effects on translation and confer hypersensitivity to paromomycin. By mimicking P. anserina mutations in the yeast Saccharomyces cerevisiae RPS15 gene, we further show that P138S and S145F induce hyperaccurate recognition of the three stop codons, whereas G139D and G139C impair UAG and UAA codon recognition. Noteworthy, in P. anserina, uS19 genetically interacts with the eRF1 and eRF3 release factors. All together, our data indicate that uS19 C-terminal tail contributes in vivo to eukaryotic translation termination, and identify key amino acids of uS19 that potentially modulate eRF1-eRF3 interaction in the pre-termination complex. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/940346v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1739f4eorg.highwire.dtl.DTLVardef@1dffb40org.highwire.dtl.DTLVardef@1e86856org.highwire.dtl.DTLVardef@11a0c02_HPS_FORMAT_FIGEXP M_FIG C_FIG Abbreviated SummaryS15/uS19 is a conserved small ribosomal protein that in eukaryotes harbors a flexible C-terminal extension proposed to interact with the A site mRNA codon during translation. Here, we describe how C-terminal variants variously affect Podospora anserina development and longevity and impact fungal ribosome and polysome formation. We reveal that stop codon recognition is significantly altered by the presence of those C-terminal variants, which either expand or on the contrary restrict termination ambiguity.

genetics↗

bioRxiv2020

A simple, cheap, and robust protocol for the identification of mating type in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an exceptional genetic system, with genetic crosses facilitated by its ability to be maintained in haploid and diploid forms. Such crosses are straightforward if the mating type/ploidy of the strains are known. Several techniques can be used to determine mating type (or ploidy), but all have limitations. Here we validate a simple, cheap and robust method to rapidly identify S. cerevisiae mating types. When cells of opposite mating type are mixed in liquid media, they "creep" up the culture vessel sides, a phenotype that can be easily detected visually. In contrast, mixtures of the same mating type or with a diploid simply settle out. The phenotype is robust to different media, cell densities, temperatures and strains, and is observable for several days. Microscopy suggests that cell aggregation during mating is responsible for the phenotype. Yeast knockout collection analysis identified 107 genes required for the creeping phenotype, with these being enriched for mating-specific genes. Surprisingly, the RIM101 signalling pathway was strongly represented. We propose that RIM101 signalling regulates aggregation as part of a wider, previously-unrecognized role in mating. The simplicity and robustness of this method makes it ideal for routine verification of S. cerevisiae mating type, with future studies required to verify its molecular basis.

microbiology↗