Role of a 300-Kilodalton Nuclear Complex in the Maturation of Trypanosoma brucei Initiator Methionyl-tRNA

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Abstract
TRNAs are transcribed as precursors containing 5′ leader and 3′ extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNA Met in Trypanosoma brucei , an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5′ leader of initiator tRNA Met precursors. One of the subunits of the complex ( T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA Met containing 5′ extensions but processed 3′ ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA Met molecules with 5′ and 3′ extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA Met . Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA Met , 3′ trimming precedes 5′ processing.