The Presence of N6‐methyladenine and 5‐methylcytosine in Trypanosoma brucei RNA

Abstract

Trypanosoma brucei is the parasite responsible for Human African Trypanosomiasis, commonly referred to as African Sleeping Sickness. In T. brucei there is a lack of promoters and other regulatory sequences normally present in DNA. Thus, posttranscriptional RNA alterations such as methylation may be important in this organism. To investigate the occurrence of RNA base methylation, a series of ELISAs for N6‐methyladenine (N6‐MeA) and 5‐methylcytosine (5‐MeC) were performed. The presence of N6‐MeA in total RNA and small RNA was detected with minimal differences between procyclic and bloodstream forms. ELISAs identified 5‐MeC in both procyclic and bloodstream form total RNA samples, and low 5‐MeC levels in small RNA. Using a robust bisulfite sequencing technique, total RNA and small RNA 5‐MeC residues were mapped with base resolution. There was an elevated frequency of 5‐MeC in tRNA with numerous residues having a level of methylation greater than 50%. To investigate the source of 5‐MeC in RNA, BLAST identified seven putative T. brucei cytosine RNA methyltransferases (TbCRMTs). Of the seven TbCRMT RNAi lines generated, 2 of the 7 resulted in growth defects when the RNAi system was activated. To study the TbCRMTs in vitro, recombinant pET28‐TbCRMT plasmids were introduced into E. coli. The TbCRMTs were expressed as N‐terminal 6x‐his tagged proteins and characterized by SDS‐PAGE and western blot analysis. The TbCRMTs are soluble and, in the future, successful protein purification will enable in vitro RNA methylation assays. In summary, the data indicate that T. brucei transcriptome contains modified RNA bases, and we have identified the 5‐MeC positions and putative enzymes responsible.Support or Funding InformationNIH 1R15AI13342801This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.