TY - JOUR
T1 - A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs
AU - Takagaki, Y.
AU - Manley, J. L.
AU - MacDonald, C. C.
AU - Wilusz, J.
AU - Shenk, T.
PY - 1990
Y1 - 1990
N2 - 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-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.
AB - 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-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.
KW - Multisubunit factor CstF
KW - mRNA processing
KW - polyadenylation
UR - http://www.scopus.com/inward/record.url?scp=0025695149&partnerID=8YFLogxK
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U2 - 10.1101/gad.4.12a.2112
DO - 10.1101/gad.4.12a.2112
M3 - Article
C2 - 1980119
AN - SCOPUS:0025695149
SN - 0890-9369
VL - 4
SP - 2112
EP - 2120
JO - Genes and Development
JF - Genes and Development
IS - 12 A
ER -