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Published on: June 15, 2018
Partial characterization of the auxiliary factors involved in apolipoprotein B mRNA editing through APOBEC-1 affinity
1Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642, USA.
Abstract:
APOBEC-1-catalyzed apolipoprotein B (apoB) mRNA editing requires auxiliary factors, but the number and functions of these factors are unknown. We have partially purified the editing activity from extracts of a McArdle cell line overexpressing His6-hemagglutinin-tagged, rat APOBEC-1 using metal-chelating affinity chromatography. The 1,200-fold purification achieved by this approach was partially dependent on exogenously added RNA containing a mooring sequence for editosome assembly. Affinity-purified editing activity could be separated by 300 mM NaCl extraction into two fractions, a salt-resistant fraction (editing fraction 1; EF1) and a salt-soluble fraction (EF2). Neither EF1 nor EF2 alone could edit apoB RNA, but when added together they reconstituted full editing activity. Previously identified candidate auxiliary factors including the p66/p44 apoB RNA binding proteins and the presumptive editosome assembly factor p240 were all present in the affinity-purified editing complex. Moreover, virtually all of p66, p240, and APOBEC-1 were present in EF1, whereas p44 was quantitatively recovered in EF2. This is the first demonstration that p66 and p44 can bind to apoB RNA independently of one another. In addition, 100- and 55-kDa apoB RNA cross-linking proteins have been identified in the APOBEC-1 affinity-purified material. RNA competition studies demonstrated that p100, p66, and p55 bound selectively to apoB RNA, whereas p44 had general RNA cross-linking characteristics. The data underscore the multiplicity of auxiliary factors potentially involved in apoB RNA editing and suggest an editosome far more complicated than may have been previously appreciated.
Insights
Researchers identified multiple auxiliary factors essential for apolipoprotein B (apoB) mRNA editing. These factors, including p66 and p44 RNA binding proteins, are crucial for the APOBEC-1 editing complex function.
Area of Science:
- Molecular Biology
- RNA Metabolism
- Protein Biochemistry
Background:
- Apolipoprotein B (apoB) mRNA editing is a critical post-transcriptional modification.
- This process requires the enzyme APOBEC-1 and several auxiliary factors.
- The precise number and functions of these auxiliary factors remain largely uncharacterized.
Purpose of the Study:
- To partially purify and characterize the auxiliary factors involved in APOBEC-1-catalyzed apoB mRNA editing.
- To elucidate the roles of specific proteins, such as p66 and p44, in the editing complex.
- To investigate the assembly and composition of the apoB RNA editing machinery.
Main Methods:
- Partial purification of APOBEC-1 editing activity from cell extracts using metal-chelating affinity chromatography.
- Fractionation of the purified complex using high salt concentrations (300 mM NaCl) into salt-resistant (EF1) and salt-soluble (EF2) components.
- Identification of associated proteins via Western blotting and RNA cross-linking assays.
- RNA competition studies to assess protein-RNA binding specificities.
Main Results:
- A 1,200-fold purification of apoB mRNA editing activity was achieved.
- The editing activity was reconstituted by combining two fractions (EF1 and EF2), indicating the presence of essential co-factors in each.
- Key proteins including p66, p44, p240, and APOBEC-1 were identified in the purified complex.
- p66 and p240 were primarily in EF1, while p44 was in EF2, demonstrating their independent binding to apoB RNA.
- Additional proteins (p100, p55) were identified, with p100, p66, and p55 showing selective binding to apoB RNA.
Conclusions:
- APOBEC-1-catalyzed apoB mRNA editing involves a complex multi-protein machinery.
- The study identified and partially characterized several auxiliary factors, including p66 and p44, which play distinct roles in the editing process.
- The findings suggest a more intricate editosome structure than previously anticipated, highlighting the complexity of RNA editing regulation.
