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The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity
M L Hayman1, M M Miller, D M Chandler
1Department of Microbiology and Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, 138 Farber Hall, Buffalo, NY 14214, USA.
Abstract:
RBP16 is a guide RNA (gRNA)-binding protein that was shown through immunoprecipitation experiments to interact with approximately 30% of total gRNAs in Trypanosoma brucei mitochondria. To gain insight into the biochemical function of RBP16, we used affinity chromatography and immunoprecipitation to identify RBP16 protein binding partners. By these methods, RBP16 does not appear to stably interact with the core editing machinery. However, fractionation of mitochondrial extracts on MBP-RBP16 affinity columns consistently isolated proteins of 12, 16, 18 and 22 kDa that were absent from MBP control columns. We describe here our analysis of one RBP16-associated protein, p22. The predicted p22 protein has significant sequence similarity to a family of multimeric, acidic proteins that includes human p32 and Saccharomyces cerevisiae mam33p. Glutaraldehyde crosslinking of recombinant p22 identified homo-multimeric forms of the protein, further substantiating its homology to p32. We confirmed the p22-RBP16 interaction and demonstrated that the two proteins bind each other directly by ELISA utilizing recombinant p22 and RBP16. p32 family members have been reported to modulate viral and cellular pre-mRNA splicing, in some cases by perturbing interaction of their binding partners with RNA. To determine whether p22 similarly affects the gRNA binding properties of RBP16, we titrated recombinant p22 into UV crosslinking assays. These experiments revealed that p22 significantly stimulates the gRNA binding capacity of RBP16. Thus, p22 has the potential to be a regulatory factor in T.brucei mitochondrial gene expression by modulating the RNA binding properties of RBP16.
Insights
Researchers identified p22, a protein that directly binds RBP16 (RNA-binding protein 16) and enhances its guide RNA binding. This interaction suggests p22 regulates gene expression in Trypanosoma brucei mitochondria.
Area of Science:
- Molecular Biology
- Biochemistry
- Parasitology
Background:
- RBP16 (RNA-binding protein 16) binds guide RNAs in Trypanosoma brucei mitochondria.
- Its precise biochemical function and interacting partners remain largely uncharacterized.
Purpose of the Study:
- To identify RBP16-associated proteins and elucidate their functional relationship.
- To investigate the role of RBP16-associated protein p22 in modulating RBP16's RNA-binding activity.
Main Methods:
- Affinity chromatography and immunoprecipitation to identify RBP16 binding partners.
- Glutaraldehyde crosslinking and ELISA to confirm protein interactions.
- UV crosslinking assays to assess p22's effect on RBP16's guide RNA binding.
Main Results:
- RBP16 does not appear to stably interact with the core editing machinery.
- A 22 kDa protein (p22) was consistently isolated with RBP16.
- p22 directly binds RBP16 and significantly enhances its guide RNA binding capacity.
Conclusions:
- p22 is a novel RBP16-binding protein in T. brucei mitochondria.
- p22 acts as a regulatory factor, modulating RBP16's RNA binding properties.
- This interaction has implications for regulating mitochondrial gene expression in T. brucei.