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Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania ( Viannia ) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 ( Tb AGO1). The five genes are conserved in Leishmania ( Viannia ) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major . In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania ( Viannia ) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′- O -methyltransferases, is required for methylation of siRNAs. Loss of Tb HEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1 −/− parasites are single stranded and associated with Tb AGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein Tb HEN1 methylates siRNA 3′ ends after they are loaded into Tb AGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of Tb HEN1 in Leishmania ( Viannia ) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.

Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1 , encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with α-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei . Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

Sequencing of a library of small RNAs from Giardia intestinalis identified a novel class of small sense and antisense RNAs homologous to the retroposon family GilT/Genie1 that is located at certain telomeres. These small RNAs may contribute to silencing GilT expression via the RNA interference pathway.

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.