Identification of target RNAs associated with DRBD18 and its methyl mutants
Arginine methylation of RNA-binding proteins (RBPs) plays a crucial role in the post-transcriptional regulation of gene expression in Trypanosoma brucei, the causative agent of African trypanosomiasis. This protozoan has evolved intricate mechanisms to regulate gene expression at the mRNA level, allowing it to adapt to diverse environmental conditions encountered during its complex life cycle. The process of arginine methylation involves the addition of methyl groups to specific arginine residues within RNA-binding domains of proteins. This modification can profoundly impact the binding affinity of RBPs to their target mRNAs, thereby influencing mRNA stability, localization, and translation efficiency. DRBD18 is one of the RNA-binding proteins (RBPs) in T. brucei that undergoes arginine methylation and has been involved in various aspects of mRNA metabolism and post-transcriptional gene regulation. T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, modulate the function of DRBD18 by arginine methylation. Studies have shown that arginine methylation of DRBD18 can modulate its interaction with target mRNAs, leading to differential regulation of gene expression. This study aims to identify the in vivo transcriptome-wide binding of DRBD18 in TBPRMT1 knockdown cells, which leads to decipher the specific mRNA targets, providing insights into the regulatory pathways modulated by this RBP. Overall design: To find out the direct targets of DRBD18 and its methyl mutants (R?F and R?K) as well as hypomethylated DRDB18 in TbPMT1 RNAi cell lines, we used a doxycycline-inducible system to overexpress these N-terminal MYC tagged DRBD18, R?F and R?K in DRBD18 RNAi cell line. Three biological replicates of each cell line in induced condition were grown for 36 hr (DRBD18 constructs) and 48 hours (TBPRMT1 knockdown). RNA immunoprecipitation was done using an anti-MYC antibody for the DRBD18 WT and methyl mutants and an anti-DRBD18 antibody for the PRMT1 experiments and RNA was isolated from Input and RIP samples and sent for sequencing.