Identification of Putative RNA Methyltransferases in Trypanosoma brucei

Abstract

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 InformationNIH grant 1R15AI133428‐01 to KTM