CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3' Untranslated Region, Altering Polysome Association and Stress Tolerance in .

In , mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3' untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the 3'-UTR probe. A second, homologous protein, Gis2, was identified in the genome of and also bound the 3'-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The 3' UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both and resulted in a modest increase in basal stability of the mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The Δ mutant and Δ Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the Δ and Δ Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis. Stress adaptation is fundamental to the success of as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a element within a G-quadruplex structure that binds two homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.