The function of the protein phosphatase Glc7p in transcription termination, RNA processing and transcriptional regulation of ribosomal protein genes

Gene transcription in general can be subdivided into three main phases: transcription initiation, elongation and termination. The enzyme that accomplishes transcription of protein coding genes, snRNAs and snoRNAs is RNA polymerase II (RNAP II). During transcription, the nascent RNA is processed in several ways in order to generate a mature functional RNA. For this, the transcripts of protein coding genes are capped at the 5’ end, introns are spliced out and the 3’ ends are processed by endonucleolytic cleavage at the poly(A) site followed by poly(A) tail synthesis (polyadenylation). In yeast, the cleavage and polyadenylation reaction requires a 3’ end processing complex consisting of the cleavage and polyadenylation factor (CPF), cleavage factor IA (CF IA), cleavage factor IB (CF IB) and the poly(A) binding protein. In contrast to pre-mRNAs, most pre-snoRNAs are processed only at their 3’ end. Furthermore, snoRNAs are not polyadenylated. CPF is not only involved in 3’ end processing, but distinct subunits of CPF have additional functions in transcription elongation and termination of mRNAs and snoRNAs. In recent years affinity purification of the CPF complex has lead to the identification of several new subunits of CPF (Ohnacker et al., 2000). Among them is the essential protein phosphatase Glc7p, the yeast homologue of mammalian protein phosphatase(PP1). Glc7p has diverse cellular functions (Stark, 1996). The specificity of a reaction that requires Glc7p is accomplished by targeting or regulatory factors that direct Glc7p to the location of the reaction or regulate its activity. The aim of this thesis was to study the function of Glc7p as part of CPF. In Chapterwe show, that Glc7p is required for the polyadenylation but not for the cleavage step of pre-mRNA 3’ end processing in vitro and in vivo. In addition, Glc7p is needed for correct poly(A) site selection. Glc7p physically interacts with several subunits of CPF and CF IA. One of them, the CPF subunit Pta1p, has been reported to be dephosphorylated by Glc7p (He and Moore, 2005). Dephosphorylation of Pta1p stimulates the polyadenylation reaction. Thus, Glc7p regulates polyadenylation via the phosphorylation state of Pta1p (He and Moore, 2005). We also observed that in glc7 mutant strains, several subunits of CPF are underrepresented. This might indicate that the activity of Glc7p is required for the formation of stable CPF complexes. Analysis of several glc7 mutants also revealed that Glc7p is involved in
transcription termination of snoRNAs (Chapter 3). Our data suggest that Glc7p
functions in the Nrd1 complex-dependent pathway of snoRNA transcription
termination. However, none of the Nrd1 complex subunits was found to be a target for
dephosphorylation by Glc7p. In contrast, Glc7p is not involved in transcription
termination of pre-mRNAs.
A reduction in poly(A)-dependent pausing in glc7 mutants indicated that
Glc7p might also be involved in regulating transcription elongation (Chapter 4).
Further investigation showed that Glc7p genetically interacts with the transcription
elongation factors Spt4p, Leo1p and Rtf1p. In addition, several glc7 mutants are
sensitive to the drug 6-azauracil (6AU). Sensitivity to 6AU is a phenotypic landmark
of transcription elongation mutants. Interestingly, the snoRNA transcription
termination defect observed in glc7 mutants is suppressed in glc7/spt4, glc7/leo1 and
glc7/rtf1 double mutants. This suggests that Glc7p acts as a factor required for
snoRNA transcription termination that modifies transcription elongation factors to
facilitate transcription termination. Therefore, Glc7p might couple transcription
elongation to transcription termination.
Microarray analysis of the temperature sensitive glc7-12 allele (Chapter 5)
indicated that Glc7p is involved in transcription regulation of ribosomal protein (RP)
and Ribi genes. Two signaling pathways control the transcription of RP and Ribi
genes in response to environmental conditions: the target of rapamycin (TOR) and the
Ras/PKA signaling pathway. These pathways regulate the localization of the
transcription factors Fhl1p, Ifh1p, Crf1p and Sfp1p to RP or Ribi gene promoters.
Epistasis experiments suggest that Glc7p acts downstream of the signaling component
PKA to regulate the transcription of RP genes (Chapter 6). In addition, we found that
Glc7p controls the nuclear localization of Yak1p and Crf1p. Yak1p is a downstream
target of the kinase PKA. Crf1p in turn is phosphorylated by Yak1p, shuttles to the
nucleus and represses transcription of RP genes. Regulation of the localization of the
co-repressor Crf1p by Glc7 could represent one of several redundant ways to suppress
transcription of RP genes.