Reconstitution of CF IA from Overexpressed Subunits Reveals Stoichiometry and Provides Insights into Molecular Topology
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Yeast cleavage factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3' end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. On the basis of our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.
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An ipa1 TS mutant was impaired for in vitro mRNA cleavage and polyadenylation and showed widespread defects in mRNA processing accuracy and efficiency with a significant bias towards the use of downstream polyadenylation sites.
“Indeed, as shown previously for TS mutants in components of CF IA and CPF complexes (20), such as pcf11 and cft2 TS mutants, an ipa1 TS mutant was impaired for in vitro mRNA cleavage and polyadenylation (fig. S8) and showed widespread defects in mRNA processing accuracy and efficiency with a significant bias towards the use of downstream polyadenylation sites (P < 2 × 10−16, Wilcoxon rank sum; Fig”
From A global genetic interaction network maps a wiring diagram of cellular function (2016) · cites this paper as [20]
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- Authors
- Gordon, J. M. B., Shikov, S., Kuehner, J. N., Liriano, M., Lee, E., Stafford, W., Poulsen, M. B., Harrison, C., Moore, C., & Bohm, A.
- Affiliations
- Tufts University (US)
- Boston Biomedical Research Institute, 64 Grove St., Watertown, Massachusetts 02472, United States
- Boston Biomedical Research Institute (US)
- Corresponding author
- Bohm, Andrew
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2011-11-02
- Volume
- 50
- Issue
- 47
- Pages
- 10203-10214
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- PMC
- PMC3234886
- OpenAlex
- W2170955600
- Cited by
- 33
- References
- 47
- Citations by year
- 2025: 1 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 5 · 2020: 1 · 2019: 2 · 2018: 2 · 2017: 3 · 2016: 3 · 2014: 4 · 2013: 1 · 2012: 4
- FWCI
- 1.17
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM041752, R01GM065972, GM65972, K12GM074869, GM41752, R25GM066567)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- cleavage factor I, Hrp1, Pcf11, Clp1, stoichiometry, protein complex reconstitution, molecular topology, mRNA cleavage, polyadenylation, yeast
- MeSH terms
- Recombinant Proteins, Saccharomyces cerevisiae, Gene Expression, Protein Subunits, Saccharomyces cerevisiae Proteins, mRNA Cleavage and Polyadenylation Factors, Protein Multimerization
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