ABSTRACT
The yeast Paf1 complex (Paf1C: Paf1, Cdc73, Ctr9, Rtf1, and Leo1) is associated with RNA Polymerase II (Pol II) at promoters and coding regions of transcriptionally active genes, but transcript abundance for only a small subset of genes is altered by loss of Paf1. By using conditional and null alleles of PAF1 and microarrays, we determined the identity of both primary and secondary targets of the Paf1C. Neither primary nor secondary Paf1C target promoters were responsive to loss of Paf1. Instead, Paf1 loss altered poly(A) site utilization of primary target genes SDA1 and MAK21, resulting in increased abundance of 3'-extended mRNAs. The 3'-extended MAK21 RNA is sensitive to nonsense-mediated decay (NMD), as revealed by its increased abundance in the absence of Upf1. Therefore, although the Paf1C is associated with Pol II at initiation and during elongation, these critical Paf1-dependent changes in transcript abundance are due to alterations in posttranscriptional processing.
Subject(s)
Nuclear Proteins/metabolism , RNA Polymerase II/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Macromolecular Substances/metabolism , Nuclear Proteins/genetics , RNA, Messenger/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription, GeneticABSTRACT
We have previously described an alternative form of RNA polymerase II in yeast lacking the Srb and Med proteins but including Pafl, Cdc73, Hprl, and Ccr4. The Pafl-RNA polymerase II complex (Paf1 complex) acts in the same pathway as the Pkc1-mitogen-activated protein kinase cascade and is required for full expression of many cell wall biosynthetic genes. The expression of several of these cell integrity genes, as well as many other Paf1-requiring genes identified by differential display and microarray analyses, is regulated during the cell cycle. To determine whether the Paf1 complex is required for basal or cyclic expression of these genes, we assayed transcript abundance throughout the cell cycle. We found that transcript abundance for a subset of cell cycle-regulated genes, including CLN1, HO, RNR1, and FAR1, is reduced from 2- to 13-fold in a paf1delta strain, but that this reduction is not promoter dependent. Despite the decreased expression levels, cyclic expression is still observed. We also examined the possibility that the Paf1 complex acts in the same pathway as either SBF (Swi4/Swi6) or MBF (Mbp1/Swi6), the partially redundant cell cycle transcription factors. Consistent with the possibility that they have overlapping essential functions, we found that loss of Paf1 is lethal in combination with loss of Swi4 or Swi6. In addition, overexpression of either Swi4 or Mbp1 suppresses some paf1delta phenotypes. These data establish that the Paf1 complex plays an important role in the essential regulatory pathway controlled by SBF and MBF.
