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The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary
Y Takagaki1, C C MacDonald, T Shenk
1Department of Biological Sciences, Columbia University, New York, NY 10027.
Abstract:
Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted. An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs.
Insights
Researchers identified the cDNA for the 64-kDa subunit of human cleavage stimulation factor, a key protein in pre-mRNA processing. This subunit
Area of Science:
- Molecular Biology
- RNA Processing
- Gene Expression
Background:
- Cleavage stimulation factor (CstF) is essential for 3'-end cleavage of mammalian pre-mRNAs.
- CstF comprises 77, 64, and 50 kDa subunits.
- The 64 kDa subunit binds RNA in a polyadenylylation signal-dependent manner.
Purpose of the Study:
- To isolate cDNAs encoding the 64 kDa subunit of human CstF.
- To characterize the RNA-binding properties of the 64 kDa subunit.
Main Methods:
- cDNA isolation and cloning of the 64 kDa CstF subunit.
- Expression of the cloned subunit in Escherichia coli.
- RNA binding assays using expressed protein fragments.
Main Results:
- cDNAs for the human 64 kDa CstF subunit were successfully isolated.
- The 64 kDa subunit possesses an N-terminal RNA binding domain and a C-terminal repeat structure.
- An N-terminal fragment containing the RNA binding domain binds RNA independently of the polyadenylylation signal.
Conclusions:
- The N-terminal RNA binding domain of the 64 kDa CstF subunit is directly involved in pre-mRNA binding.
- Structural analysis predicts a unique alpha-helical repeat region within the C-terminus.