Integrative genome-wide analysis reveals the RNA-binding motifs of yeast Pcf11 and its role in fine-tuning terminator strength
Pcf11 is a transcriptional termination factor that interferes with cell survival and mRNA 3'-end processing. However, the mechanism by which Pcf11 regulates transcriptional termination and the molecular basis of its interaction with the terminator remain unclear. Here, we performed gene expression profile, binding pattern, and RNA-binding motif studies on Pcf11. We used in vivo CLIP-seq to map thousands of Pcf11 binding sites in Saccharomyces cerevisiae and revealed the RNA-binding motifs of Pcf11 in the 3'-UTR region. Overall design: The complete structural domains are essential for Pcf11 to perform its functions, C-terminal C2HC zinc finger defects can interfere with cell survival and influence mRNA 3'-end processing. The ?Pcf11 and Pcf11-AARR strains were constructed by directly deleting Pcf11 from the genome and mutating four conserved amino acid residues (Y575A/E577R/E578R/W582A) to investigate in-depth the biological function of Pcf11. In addition, overexpressing Pcf11 with a strong promoter (TEF1p) and terminator (GRS1t) or a weak promoter (CDC60p) and terminator (DITIt) resulted in transcription fluctuation. RNA-seq was conducted to compare the global gene expression patterns between the WT, ?Pcf11,Pcf11-AARR, Pcf11-OE1 and Pcf11-OE2 strains to investigate the effect of Pcf11 deletion on transcription.