Impact of overexpression of DRBD18 and its methyl mutants on the gene expression pattern of T. brucei: A transcriptomic approach
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. Studies have shown that arginine methylation of DRBD18 can modulate its interaction with target mRNAs, leading to differential regulation of gene expression. Here, we have done a comprehensive transcriptomic analysis of T. brucei overexpressing DRBD18 and its methyl mutants R->F (methylmimic) and R->K (hypomethylated), aiming to elucidate the regulatory mechanisms orchestrated by DRBD18 and its potential impact on the parasite's gene expression landscape. We identified specific mRNA targets exhibiting differential expression patterns in response to DRBD18, R->F and R->K overexpression, providing insights into the regulatory pathways modulated by this RBP. Overall design: To find out the impact of overexpression of DRBD18 and its methyl mutants (R->F and R->K), we generated two methyl mutants. The first one is DRBD18(R->F), here we mutated the three identified methyl arginines to phenylalanine, creating a methyl mimic DRBD18. In the second methyl mutant, DRBD18(R->K), we replaced the methyl arginine with lysine. We used a doxycycline-inducible system to overexpress these N-terminal MYC tagged DRBD18, R->F and R->K. Three biological replicates of each cell line in the presence and absence of doxycycline, grown for 36 hr. have been used to isolate RNA for RNA sequencing.