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.

[1]  J. Manley,et al.  A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates , 1990, Molecular and cellular biology.

[2]  M. Wickens,et al.  The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase , 1990, Molecular and cellular biology.

[3]  T. Maniatis,et al.  Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus , 1990, Nature.

[4]  J. Nevins,et al.  An ordered pathway of assembly of components required for polyadenylation site recognition and processing. , 1989, Genes & development.

[5]  G. Dreyfuss,et al.  A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts , 1989, The Journal of cell biology.

[6]  J. Manley,et al.  Four factors are required for 3'-end cleavage of pre-mRNAs. , 1989, Genes & development.

[7]  J. Manley,et al.  Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation , 1989, Molecular and cellular biology.

[8]  S. Jacob,et al.  Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor , 1989, Molecular and cellular biology.

[9]  G. Christofori,et al.  Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro , 1989, Molecular and cellular biology.

[10]  C. Moore,et al.  Two proteins crosslinked to RNA containing the adenovirus L3 poly(A) site require the AAUAAA sequence for binding. , 1988, The EMBO journal.

[11]  T. Shenk,et al.  The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites , 1988, Molecular and cellular biology.

[12]  G. Christofori,et al.  3′ cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP , 1988, Cell.

[13]  N. Proudfoot,et al.  A beginning to the biochemistry of polyadenylation. , 1988, Trends in genetics : TIG.

[14]  N. Acheson,et al.  A rabbit beta‐globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA. , 1988, The EMBO journal.

[15]  C. Moore,et al.  Components required for in vitro cleavage and polyadenylation of eukaryotic mRNA. , 1988, Nucleic acids research.

[16]  Y. Osheim,et al.  Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. , 1988, Genes & development.

[17]  J. Manley,et al.  Polyadenylation of mRNA precursors. , 1988, Biochimica et biophysica acta.

[18]  J. E. Stefano,et al.  Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro , 1988, Molecular and cellular biology.

[19]  J. Manley,et al.  A functional mRNA polyadenylation signal is required for transcription termination by RNA polymerase II. , 1988, Genes & development.

[20]  J. Manley,et al.  Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation , 1988, Cell.

[21]  G. Dreyfuss,et al.  Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. , 1988, Trends in biochemical sciences.

[22]  T. Shenk,et al.  A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif , 1988, Cell.

[23]  C. Moore,et al.  Sedimentation analysis of polyadenylation-specific complexes , 1988, Molecular and cellular biology.

[24]  T Humphrey,et al.  Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3′ processing complexes. , 1987, The EMBO journal.

[25]  J E Darnell,et al.  A poly(A) addition site and a downstream termination region are required for efficient cessation of transcription by RNA polymerase II in the mouse beta maj-globin gene. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Wickens,et al.  Specific pre‐cleavage and post‐cleavage complexes involved in the formation of SV40 late mRNA 3′ termini in vitro. , 1987, The EMBO journal.

[27]  C. Cole,et al.  Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA , 1987, Molecular and cellular biology.

[28]  C. Moore,et al.  Electrophoretic separation of polyadenylation-specific complexes. , 1987, Genes & development.

[29]  M. Wickens,et al.  Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs , 1987, Molecular and cellular biology.

[30]  N. Proudfoot,et al.  Alpha‐thalassaemia caused by a poly(A) site mutation reveals that transcriptional termination is linked to 3′ end processing in the human alpha 2 globin gene. , 1986, The EMBO journal.

[31]  C. Moore,et al.  Analysis of RNA cleavage at the adenovirus‐2 L3 polyadenylation site. , 1986, The EMBO journal.

[32]  M. Wickens,et al.  The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro , 1986, Molecular and cellular biology.

[33]  N. Ringertz,et al.  Intranuclear localization of snRNP antigens , 1986, The Journal of cell biology.

[34]  P. Sharp,et al.  Accurate cleavage and polyadenylation of exogenous RNA substrate , 1985, Cell.

[35]  J. Manley,et al.  RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro , 1985, Molecular and cellular biology.

[36]  G. Dreyfuss,et al.  Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells , 1984, The Journal of cell biology.

[37]  G. Dreyfuss,et al.  Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies , 1984, Molecular and cellular biology.

[38]  S. Goodbourn,et al.  α-Thalassaemia caused by a polyadenylation signal mutation , 1983, Nature.

[39]  K. Hancock,et al.  India ink staining of proteins on nitrocellulose paper. , 1983, Analytical biochemistry.

[40]  J. Manley.  Accurate and specific polyadenylation of mRNA precursors in a soluble whole-cell lysate , 1983, Cell.

[41]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[42]  P. Sharp,et al.  Rna synthesis in isolated nuclei processing of adenovirus serotype 2 late messenger rna precursors. , 1982, Journal of molecular biology.

[43]  J. Darnell,et al.  Transcription and accurate polyadenylation in vitro of RNA from the major late adenovirus 2 transcription unit , 1982, Cell.

[44]  R. Hawkes,et al.  A dot-immunobinding assay for monoclonal and other antibodies. , 1982, Analytical biochemistry.

[45]  J. Nevins,et al.  Nuclear RNA is spliced in the absence of poly(A) addition , 1981, Cell.

[46]  J. Steitz,et al.  Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[47]  T. Shenk,et al.  The sequence 5′-AAUAAA-3′ forms part of the recognition site for polyadenylation of late SV40 mRNAs , 1981, Cell.

[48]  C. Merril,et al.  Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. , 1981, Science.

[49]  J. Darnell,et al.  Order of polyadenylic acid addition and splicing events in early adenovirus mRNA formation , 1980, Journal of virology.

[50]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[51]  J. Nevins,et al.  Steps in the processing of Ad2 mRNA: Poly(A)+ Nuclear sequences are conserved and poly(A) addition precedes splicing , 1978, Cell.

[52]  N. Proudfoot,et al.  3′ Non-coding region sequences in eukaryotic messenger RNA , 1976, Nature.

[53]  W. Hunter,et al.  The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. , 1973, The Biochemical journal.

[54]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.