Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22

摘要

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3’ processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1 Δ, swd22 Δ and dis2 Δ cells, which included the DPS Δ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640–710. A TFIIS-like domain (aa 173–330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22. Author summary Multi-layered regulatory inputs to eukaryal gene expression target the initiation, elongation, and termination steps of RNA polymerase II transcription. Termination is coupled to, and ensues from, the 3’ processing of nascent RNA by multi-protein assemblies. The fission yeast Cleavage and Polyadenylation Factor complex, CPF, is an exemplary 3’ processing machine composed of 13 protein subunits, three of which (named Dis2, Ppn1, and Swd22) form a DPS sub-complex within CPF. In this study, we interrogated the effects of DPS null mutations on the fission yeast transcriptome. We probed the physical interactions of Ppn1 and its genetic interactions with other components of the transcription and RNA processing machinery. We delineated distinct binding sites within Ppn1 for Dis2 and Swd22 and then showed that perturbation of these sites interdicts Ppn1’s essential activities in vivo.