Pin4 Links Post-transcriptional and Transcriptional Responses to Glucose Starvation in Yeast
Adaptation to environmental change is essential in all organisms, with RNA-binding proteins (RBPs) playing critical roles in rapid cellular responses. We analyzed the largely uncharacterized yeast RBP Pin4, and its involvement in adaptation to glucose depletion. A UV crosslinking technique to identify protein-RNA interactions (reCRAC) revealed that in glucose conditions Pin4 selectively binds a specific motif in 3' UTRs of mRNAs involved in glycolysis, amino acid, and mitochondrial metabolism. Following glucose withdrawal, Pin4-RNA binding was greatly reduced, with residual binding favoring transcripts associated with protein translation. Cells lacking Pin4 were greatly impaired in recovery from nutrient starvation and hypersensitive to oxidative stress, consistent with the mRNA targets. RNAseq and reporter assays indicated that loss of Pin4 caused increased target mRNA abundance. In wildtype yeast, glucose depletion induces diauxic shift, with massive changes in transcription patterns. Very unexpectedly, this response was almost entirely abolished in cells lacking Pin4, or carrying a point mutation in its RNA-recognition motif. We conclude that Pin4 contributes to energy homeostasis by regulating post-transcriptional and transcriptional responses, and postulate that this key stress response pathway is riboregulated. Overall design: To investigate the role of yeast RNA binding protein Pin4, we created N-terminal FLAG-Ala4-His8 (FH) tagged Pin4 strain in BY4741 background  to identify by reCRAC the RNAs bound by Pin4 during growth in 2% glucose medium (glucose) and following transfer to 2% glycerol plus ethanol (Gly/EtOH) medium for 16 min (stress). We also deleted PIN4 and did RNAseq in glucose conditions and in shifting the cells to 2% Gly/EtOH for 16 min to determine whether the loss of Pin4 affects target mRNA abundance.