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.

Human African Trypanosomiasis (African sleeping sickness) is a potentially fatal disease in sub‐Saharan Africa that is caused by the protozoan parasite Trypanosoma brucei. This vector‐borne disease afflicts tens of thousands of individuals every year, and without a vaccine, it is a prominent threat to the health and safety of many African communities. RNA methylation may be an important mechanism for the regulation of gene expression in T. brucei, as this organism lacks regulatory DNA sequences. The presence of 5‐methylcytosine has been detected in the RNA of T. brucei through sodium bisulfite sequencing. Seven putative RNA methyltransferases were identified in the genome of the parasite via bioinformatics and were named TbCRMTs ( T. brucei cytosine RNA methyl transferases). The levels of TbCRMT RNA in both procyclic and bloodstream forms were measured via RT‐qPCR and demonstrated the expression of all seven genes in both life cycle stages. TbCRMT4 is 103 kDa, and contains the conserved SAM binding domain and catalytic cysteines found inbona fide RNA methyltransferases. TbCRMT4 is required for maximum parasite growth as determined by RNAi knockdowns. In an attempt to determine whether TbCRMT4 functions as an RNA methyltransferase, the gene was expressed in E. coli with an N‐terminal 6x‐His tag. SDS‐PAGE results suggested that the full‐length protein was fragmented due to the presence of multiple bands. In light of this, the 6x‐His tag was moved to the C‐terminus and fragments of the gene containing putative SAM‐binding and catalytic sites were then amplified by PCR. The PCR products were then inserted into the pET101/D vector and transformed into competent E. coli. Expression of each construct in E. coli was induced under different conditions, with varying degrees of success. The construct which contains amino acids 87‐852, when expressed at 37°C, is most reliably produced, as confirmed via SDS‐PAGE/Coomassie blue staining and Western Blot analysis. Isolation of this protein was attempted using denaturing protocols and His‐affinity chromatography. Solubility of the protein has proven difficult, thus optimization of protein isolation is still ongoing. Ultimately, we plan to isolate the TbCRMT4 87‐852 protein and conduct a series of methyltransferase assays, which will determine whether or not this protein catalyzes RNA methylation. By determining if TbCRMT4 functions as an RNA methyltransferase, we can further understand the T. brucei epitranscriptome. Support or Funding Information NIH grant 1R15AI133428‐01 to KTM