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Impact of overexpression of DRBD18 and its methyl mutants on the gene expression pattern of T. brucei: A transcriptomic approach

ABSTRACT: 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.

ORGANISM(S): Trypanosoma brucei

PROVIDER: GSE262448 | GEO | 2025/04/01

REPOSITORIES: GEO

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Project description:Protein methylation has been implicated in many important biological contexts including signaling, metabolism, and transcriptional control. Despite the importance of this post- translational modification, the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied due to the lack of robust, well-characterized techniques for methyl-peptidemethyl peptide enrichment. Here, to better investigate protein methylation, we optimized and compared two methods for methyl-peptidemethyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX). Comparison of these methods revealed that they are largely orthogonal for monomethyl arginine (MMA), suggesting that the usage of both techniques is required to provide a global view of protein methylation. Using both IAP and SCX, we investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1). Together, these techniques allowed us to quantify over ~1,000 arginine methylation sites on 407 proteins. Of these methylation sites, PRMT1 knockdown resulted in significant changes to 110 arginine methylation sites on 59 proteins. In contrast, zero lysine methylation sites were significantly changed upon PRMT1 knockdown. In PRMT1 knockdown cells, 24 MMA sites exhibited both significant downregulation (ie, putative PRMT1-mediated MMA targets) and 63 significant upregulation (ie, putative PRMT1-mediated ADMA targets). PRMT1 knockdown induced significant changes in asymmetric dimethyl arginine (ADMA), consistent with being PRMT1 targets. Additionally, We also observed that PRMT1 knockdown induced significant increases in symmetric dimethyl arginine (SDMA) methylation, suggesting that loss of PRMT1 activity allows scavenging of PRMT1 substrates by Type II PRMTs. Analysis of the subcellular localization and protein function of the significantly changing methyl-proteins revealed that the majority of PRMT1 substrates are localized to the nucleus and involved in RNA and DNA binding. Taken together, our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques, identify novel PRMT1 methylation targets, and highlight in order to understand the dynamic interplay between methyltransferases in mammalian cells.

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Impact of overexpression of DRBD18 and its methyl mutants on the gene expression pattern of T. brucei: A transcriptomic approach

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