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Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for
M M Kessler1, M F Henry, E Shen
1Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus.
Insights
Yeast pre-mRNA cleavage and polyadenylation require four factors. Researchers identified Hrp1 protein as a key component (CF IB), demonstrating its role in RNA binding and poly(A) tail length, linking 3'-end processing to mRNA export.
Area of Science:
- Molecular Biology
- RNA Processing
- Yeast Genetics
Background:
- Accurate pre-mRNA 3'-end cleavage and polyadenylation in yeast depend on four factors: CF I, CF II, PF I, and PAP.
- CF I is further resolvable into CF IA and CF IB subcomplexes, with the identity of CF IB previously unknown.
Purpose of the Study:
- To identify the protein component of the CF IB fraction involved in yeast pre-mRNA processing.
- To characterize the function and RNA-binding properties of the identified protein.
- To investigate the genetic interactions and cellular localization of the protein in relation to mRNA processing and export.
Main Methods:
- Protein purification and functional assays in vitro (cleavage and polyadenylation).
- Recombinant protein expression and characterization (UV cross-linking).
- Site-directed mutagenesis and genetic analysis (temperature-sensitive mutants, suppressor screens).
- Analysis of protein-protein interactions and subcellular localization (shuttling assays).
Main Results:
- The 73-kD Hrp1 protein was identified as the functional component of CF IB.
- Recombinant Hrp1p restored both cleavage and polyadenylation activity in vitro.
- Hrp1p specifically binds to RNA via its RRM motifs, requiring the (UA)6 polyadenylation efficiency element, and its binding is enhanced by CF II.
- A temperature-sensitive Hrp1 mutant produced mRNAs with shorter poly(A) tails.
- Hrp1p interacts with Rna15p and Rna14p (CF IA components) and shuttles between the nucleus and cytoplasm.
Conclusions:
- Hrp1 protein is essential for yeast pre-mRNA 3'-end cleavage and polyadenylation, functioning as CF IB.
- Hrp1p's RNA-binding activity and interactions with other processing factors highlight its central role.
- Hrp1p's nucleocytoplasmic shuttling suggests a functional link between mRNA 3'-end processing and nuclear export.