Related Experiment Videos
A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs
Y Takagaki1, J L Manley, C C MacDonald
1Department of Biological Sciences, Columbia University, New York, New York 10027.
Abstract:
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.
Insights
Researchers purified cleavage-stimulation factor (CstF), essential for accurate messenger RNA (mRNA) polyadenylation. This protein complex, with subunits of 77, 64, and 50 kD, plays a crucial role in mRNA 3'-end processing.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Accurate polyadenylation is critical for messenger RNA (mRNA) stability and translation.
- The precise molecular mechanisms governing mammalian pre-mRNA 3 -end processing remain under investigation.
Purpose of the Study:
- To purify and characterize the protein factor responsible for accurate in vitro polyadenylation of mammalian pre-mRNAs.
- To elucidate the subunit composition and RNA-binding properties of this factor.
Main Methods:
- Protein purification and characterization.
- Monoclonal antibody production and application.
- UV cross-linking assays to study RNA-protein interactions.
- Indirect immunofluorescence microscopy.
Main Results:
- Identified and purified cleavage-stimulation factor (CstF), a heterotrimeric complex (77, 64, 50 kD).
- Demonstrated CstF's requirement for efficient cleavage of polyadenylation substrates.
- Showed specific binding of CstF to AAUAAA signal sequences via its 64-kD subunit.
- Localized CstF to the nucleus in a granular pattern, distinct from splicing factors.
Conclusions:
- CstF is a key regulator of pre-mRNA polyadenylation and 3 -end processing.
- The 64-kD subunit directly interacts with the AAUAAA polyadenylation signal.
- CstF likely initiates 3 -end processing on nascent transcripts in conjunction with other factors.