Talk on Friday 04:18-04:30pm submitted by Kaylen Lott
DRBD18 is an essential Trypanosome RNA binding protein that modulates RNA abundance and is regulated by arginine methylation
Kaylen Lott (Microbiology and Immunology, SUNY at Buffalo), Shreya Mukhopadhyay (Microbiology and Immunology, SUNY at Buffalo), Jie Yao (Microbiology and Immunology, SUNY at Buffalo), Jun Li, Jun Qu (Pharmaceutical Sciences, SUNY at Buffalo), Yijun Sun (Microbiology and Immunology, SUNY at Buffalo), Laurie Read (Microbiology and Immunology, SUNY at Buffalo)
Unlike most eukaryotes, T. brucei regulates gene expression almost entirely post-transcriptionally, thus RNA binding proteins are key factors in gene regulation. In yeast and humans, RNA binding proteins constitute a significant proportion of proteins that are substrates for arginine methylation, and methylation can dramatically affect their functions. Indeed, our global proteomic screen in T. brucei revealed ~100 arginine methylproteins with known or predicted functions in RNA metabolism. DRBD18 is an RRM (RNA recognition motif)-containing protein that harbors three methylarginines positioned between its two RRMs. DRBD18 repression leads to a severe growth defect in procyclic form T. brucei. RNAseq analysis of DRBD18-repressed cells identified 417 transcripts that decrease in abundance and 568 transcripts that increase 1.8 to 40-fold upon DRBD18 depletion. To begin to understand the role of arginine methylation in DRBD18 function, we performed in vivo cross-linking immunoprecipitation (CLIP) assays in trypanosomes harboring DRBD18 mutated at the identified methylated arginines. Intriguingly, R to F methylmimic DRBD18 exhibits a substantial loss of RNA binding in vivo, while non-methylatable R to K mutants display increased RNA binding. Moreover, in complementation assays, both methylmimic and non-methylatable DRBD18 fail to fully complement growth of the DRBD18 knockdown cell line. Together, these data suggest that DRBD18 RNA binding activity is regulated by arginine methylation, and mis-regulation of its RNA binding can impact parasite growth. When these complemented cells were probed for their ability to restore RNA levels altered by repression of DRBD18, nonmethylatable and methylmimc cells displayed opposite phenotypes on RNA levels. Mass spectrometry reveals that DRBD18 methylmutants bind distinct RNA binding proteins which may influence DRBD18 function. These data are consistent with a model in which methylation modulates DRBD18 affects on protein-RNA and protein-protein interactions.
Keywords: RNA binding protein, arginine methylation, transcriptome