Characterization of the Putative Cytosine RNA Methyltransferase TbCRMT5 from Trypanosoma brucei

Abstract

RNA methylation is a type of posttranscriptional modification that plays an important role in controlling gene expression. The organism Trypanosoma brucei, the protozoan parasite responsible for Human African Trypanosomiasis, does not seem to have abundant promoter regions or transcriptional regulation machinery. Thus, RNA methylation may play an especially important role in regulating gene expression in this organism. Recently, we have identified seven putative cytosine RNA methyltransferase (CRMT) genes in T. brucei. One of the putative CRMTs, termed CRMT5, is required for maximum parasite growth. The purpose of this research is to provide evidence for genuine RNA methyltransferase activity. CRMT5 was expressed in E. coli with an N‐terminal 6x‐histidine tag and purified using a His‐affinity column. Purified CRMT5 was used in a series of methyltransferase assays using luciferase activity as a readout. CRMT5 addition resulted in luciferase activity in the presence of cytosine‐containing RNA (T. brucei total RNA and Poly‐IC RNA). There was little to no luciferase activity observed in the presence of RNA that lacks cytosine or when a mock purification from E. coli without the CRMT5 gene was used. Furthermore, a CRMT5 mutant was created changing a putative active site cysteine to alanine (C439A). We expect the mutant to show reduced luciferase activity in the methyltransferase assay. Future experiments will include performing a methyltransferase reaction with CRMT5 and subsequently isolating the RNA for bisulfite sequencing to confirm the methylation of cytosine bases, performing methyltransferase assays with different substrates to determine if there is a binding preference, and confirming our methyltransferase assay results with a radioactivity assay. Evidence for the presence of 5‐methylcytosine and RNA methyltransferases indicates the presence of a process to create an epitranscriptome in T. brucei.Support or Funding InformationNIH grant 1R15AI133428‐01 to KTM