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Arginine Methylation Modulates RNA Biology Of African Trypanosomes

Kinetoplastid parasites, including Trypanosoma brucei, T. cruzi, and Leishmania spp. share unique biological features. Prominent among these is the lack of transcriptional control. Instead, kinetoplastids regulate gene expression during development and in response to internal and external cues almost entirely by posttranscriptional processes, including mRNA stability and translation. Thus, RNA binding proteins (RBPs) are the key gene regulatory factors in these organisms. Arginine methylation is a posttranslational modification that disproportionately targets RBPs in humans, yeast, and, T. brucei, the causative agent of Human African Trypanosomiasis. To begin to define the impacts of arginine methylation on T. brucei gene expression, we focused on DRBD18, an abundant and essential protein that contains three methylarginine residues between its two RNA binding domains. DRBD18 has opposing effects on the stabilities of two different cohorts of mRNA, such that DRBD18 depletion leads to significant increases and decreases in the abundances of hundreds of mRNAs, including those encoding several kinases, phosphatases, and RBPs. By analysis of DRBD18 knockdown parasites complemented with wild type, hypomethylated (RK), or methylmimic (RF) DRBD18 we demonstrated that methylation promotes the stabilization of those mRNAs that are typically stabilized by DRBD18, whereas hypomethylated DRBD18 is crippled for this function. In direct contrast, destabilization of a different subset of mRNAs relies on hypomethylated DRBD18, and methylmimic DRBD18 cannot perform this function. DRBD18 interacts with the protein arginine methyltransferase, TbPRMT1, and TbPRMT1 knockdown parasites display phenotypes analogous to those of cells complemented with hypomethylated DRBD18, confirming regulation of DRBD18 by methylation. Our data indicate that arginine methylation impacts the association of both mRNAs and effector proteins with DRBD18. For example, RIP/qRT‐PCR revealed differential association of DRBD18(RF) and DRBD18(RK) with target mRNAs. Through tandem affinity purification and mass spectrometry, we isolated several RBPs that interact with hypomethylated or methylmimic DRBD18 in a mutually exclusive manner. Because DRBD18 target mRNAs have unusually long 3′ UTRs, this suggests that methylation regulates association of DRBD18 with other RBPs, which together lead to combinatorial effects on target mRNA fate. In addition, methylmimic and hypomethylated DRBD18 differentially interact with proteins involved in numerous aspects of RNA biology, including RNA transport, deadenylation, and translation. Together, our results demonstrate that arginine methylation acts a molecular switch towards a major trypanosome RBP, DRBD18, highlighting the power of this posttranslational modification to dramatically remodel the transcriptome.