The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing.
Saccharomyces cerevisiae Pta1 is a component of the cleavage/polyadenylation factor (CPF) 3'-end processing complex and functions in pre-mRNA cleavage, poly(A) addition, and transcription termination. In this study, we investigated the role of the N-terminal region of Pta1 in transcription and processing. We report that a deletion of the first 75 amino acids (pta1-Delta75) causes thermosensitive growth, while the deletion of an additional 25 amino acids is lethal. The pta1-Delta75 mutant is defective for snoRNA termination, RNA polymerase II C-terminal domain Ser5-P dephosphorylation, and gene looping but is fully functional for mRNA 3'-end processing. Furthermore, different regions of Pta1 interact with the CPF subunits Ssu72, Pti1, and Ysh1, supporting the idea that Pta1 acts as a scaffold to organize CPF. The first 300 amino acids of Pta1 are sufficient for interactions with Ssu72, which is needed for pre-mRNA cleavage. By the degron-mediated depletion of Pta1, we show that the removal of this essential region leads to a loss of Ssu72, yet surprisingly, in vitro cleavage and polyadenylation remain efficient. In addition, a fragment containing amino acids 1 to 300 suppresses 3'-end processing in wild-type extracts. These findings suggest that the amino terminus of Pta1 has an inhibitory effect and that this effect can be neutralized through the interaction with Ssu72.
Ghazy
Mohamed A
MA
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA.
He
Xiaoyuan
X
Singh
Badri Nath
BN
Hampsey
Michael
M
Moore
Claire
C
D002352:Carrier Proteins / Q000502:physiology*; D050505:Mutant Proteins; D010749:Phosphoprotein Phosphatases; D026723:Polyadenylation; D026721:RNA 3' End Processing; D012322:RNA Precursors / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D017384:Sequence Deletion; D014158:Transcription, Genetic; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry / Q000502:physiology*
D002352:Carrier Proteins; D050505:Mutant Proteins; C076229:PTA1 protein, S cerevisiae; D012322:RNA Precursors; D020537:RNA, Small Nucleolar; C416934:SSU72 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D010749:Phosphoprotein Phosphatases