Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'-ends.
Messenger RNA 3'-end formation is functionally coupled to transcription by RNA polymerase II. By tagging and purifying Ref2, a non-essential protein previously implicated in mRNA cleavage and termination, we isolated a multiprotein complex, holo-CPF, containing the yeast cleavage and polyadenylation factor (CPF) and six additional polypeptides. The latter can form a distinct complex, APT, in which Pti1, Swd2, a type I protein phosphatase (Glc7), Ssu72 (a TFIIB and RNA polymerase II-associated factor), Ref2, and Syc1 are associated with the Pta1 subunit of CPF. Systematic tagging and purification of holo-CPF subunits revealed that yeast extracts contain similar amounts of CPF and holo-CPF. By purifying holo-CPF from strains lacking Ref2 or containing truncated subunits, subcomplexes were isolated that revealed additional aspects of the architecture of APT and holo-CPF. Chromatin immunoprecipitation was used to localize Ref2, Ssu72, Pta1, and other APT subunits on small nucleolar RNA (snoRNA) genes and primarily near the polyadenylation signals of the constitutively expressed PYK1 and PMA1 genes. Use of mutant components of APT revealed that Ssu72 is important for preventing readthrough-dependent expression of downstream genes for both snoRNAs and polyadenylated transcripts. Ref2 and Pta1 similarly affect at least one snoRNA transcript.
Nedea
Eduard
E
Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
He
Xiaoyuan
X
Kim
Minkyu
M
Pootoolal
Jeff
J
Zhong
Guoqing
G
Canadien
Veronica
V
Hughes
Timothy
T
Buratowski
Stephen
S
Moore
Claire L
CL
Greenblatt
Jack
J
D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D026723:Polyadenylation; D011233:Precipitin Tests; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry* / Q000378:metabolism*
D002352:Carrier Proteins; D002843:Chromatin; D004274:DNA, Recombinant; C441449:Mpe1 protein, S cerevisiae; C076229:PTA1 protein, S cerevisiae; D010455:Peptides; C092121:REF2 protein, S cerevisiae; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C416934:SSU72 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D012313:RNA; C092048:GLC7 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D001616:beta-Galactosidase