A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs.
We have purified and characterized a factor required for accurate polyadenylation of mammalian pre-mRNAs in vitro. This factor, called cleavage-stimulation factor (CstF), is composed of three distinct polypeptide subunits of 77, 64, and 50 kD. Using monoclonal antibodies directed against the 64- and 50-kD subunits, we show that CstF is required for efficient cleavage of polyadenylation substrates. Furthermore, CstF present in unfractionated nuclear extracts interacts with pre-mRNAs containing the signal sequence AAUAAA, but not AAGAAA, in such a manner that the 64-kD subunit can be cross-linked to the RNA by UV light. This polypeptide is thus identical to the previously described 64-kD nuclear protein that binds to AAUAAA-containing RNAs. Finally, indirect immunofluorescence of fixed cells indicates that CstF is distributed diffusely throughout the nucleus in a granular pattern distinct from the "speckled" pattern displayed by factors involved in pre-mRNA splicing, but similar to that of heterogeneous nuclear ribonucleoproteins. A model is presented in which CstF binds specifically to nascent RNA polymerase II transcripts and, by interacting with other factors, results in a rapid initiation of 3'-end processing of pre-mRNAs.
Takagaki
Y
Y
Department of Biological Sciences, Columbia University, New York, New York 10027.
Manley
J L
JL
MacDonald
C C
CC
Wilusz
J
J
Shenk
T
T
D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism*; D011233:Precipitin Tests; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D011061:Poly A