5 prime mRNA cap modifications involved in gene expression control in T. brucei

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RNA methylation is rapidly emerging as a gene expression regulator. In Trypanosoma brucei and related parasites, seven RNA methylations are present at the 5′-end of capped mRNA, which may play a role in post-transcriptional gene expression by regulating protein synthesis. This makes the Trypanosome an outstanding target for mRNA cap methylation studies.We showed that putative RNA methyltransferase possesses a novel cap-dependent RNA methyltransferase activity. The recombinant enzyme was capable of transferring the methyl group to the capped RNA. The activity was enhanced by the presence of m7G cap and 2′-O ribose methylations. However, it does not appear to methylate the known methylation site found. We plan to determine the position and what kind of RNA modification this enzyme support by analyzing the RNA products using mass spectrometry.We expect to identify the role of the RNA methyltransferase in parasite gene expression by generating CRISPR knock-out and RNAi knockdown in T. cruzi and T.brucei, respectively. We constructed a targeting plasmid encoding for Cas9-EGFP protein and guide RNA under the control of a tetracycline-inducible T7 RNA polymerase promoter. A stable T. cruzi cell line was obtained to analyze the system's effectiveness. As an alternative and complementary approach, we also prepared targeting plasmid to deplete the RNA methyltransferase in T. brucei. Comparative, genome-wide analyses of RNA isolated from CRISPR knock-out and RNAi knockdown cell lines should provide insight into how RNA methylation plays a role in eukaryotic gene expression.