Identification of RNA Modification Enzymes in 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. We have identified the presence of 5‐methylcytosine in T. brucei RNA using both mass spectrometry and sodium bisulfite sequencing. Recently, we have identified seven putative cytosine RNA methyltransferase (CRMT) genes in T. brucei. All seven CRMTs are expressed in bloodstream and procyclic form parasites, as detected by qRT‐PCR. Two of the putative CRMTs, termed CRMT4 and CRMT5, are required for maximum parasite growth. Although we suspect these genes to be RNA methyltransferases, we do not have evidence for RNA methyltransferase activity. Both CRMT4 and CRMT5 were expressed in E. coli with N‐terminal 6x‐histidine tags. CRMT4 was produced in E. coli but was difficult to purify. SDS‐PAGE results for an N‐terminal His tagged protein indicate CRMT4 insolubility. Our next step will be to use PCR to create smaller fragments of CRMT4, which may be soluble. CRMT5 was produced in E. coli with an N‐terminal His 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 results 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. Our next step will be to perform a methyltransferase reaction with CRMT5 and subsequently isolate the RNA for bisulfite sequencing to confirm the methylation of cytosine bases. 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, 1R15AI133428‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.