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

Eduard Nedea, Xiaoyuan He, Minkyu Kim, Jeff Pootoolal, Guoqing Zhong, Veronica Canadien, Timothy Hughes, Stephen Buratowski, Claire L. Moore, Jack Greenblatt

Research output: Contribution to journalArticlepeer-review

155 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)33000-33010
Number of pages11
JournalJournal of Biological Chemistry
Volume278
Issue number35
DOIs
StatePublished - Aug 29 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of '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'. Together they form a unique fingerprint.

Cite this