Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei

Abstract

Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5-monophosphate and a blocked 3 terminus, suggesting that 3 end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35-to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen.repeat-derived siRNAs ͉ retroposons ͉ RNase III ͉ siRNA modification

Gene silencing in microalgae: Mechanisms and biological roles

“…4). In contrast, the multidomain structure of the Dicer enzymes of higher eukaryotes is not well maintained among protists (Cerutti and CasasMollano, 2006) and an RNaseIII motif might be all that is strictly required for function (Cerutti and Casas-Mollano, 2006;Patrick et al, 2009). RDR-related proteins are structurally conserved but, consistent with an ancillary role in typical RNAi (Cerutti and CasasMollano, 2006), they appear to have a fairly limited taxonomic distribution among the examined microalgae (Table 1).…”

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

“…2A; Patrick et al 2009). If the 59 ends of tRNA introns that accumulate in xrn1D cells have a terminal phosphate, CIP treatment could lead to a change in electrophoretic mobility; in contrast, TEX treatment would result in intron degradation.…”

Comparative transcriptome analysis of small noncoding RNAs in different stages of Trypanosoma brucei

“…In contrast, 5 These authors contributed equally to this work. 6 siRNAs are ubiquitous and abundant in many protozoa (Robinson and Beverley 2003;Ullu et al 2005;Zhang et al 2008;Patrick et al 2009;Lye et al 2010;Tschudi et al 2012), suggesting that siRNAs, but not miRNAs, may be the key molecules in the RNA interference (RNAi) pathway in protozoan species.…”

“…Trypanosoma brucei is one of the most ancient eukaryotes in which RNAi has been discovered and extensively studied (Patrick et al 2009). It has been reported that endo-siRNAs in T. brucei could be derived from retroposons and repeat sequences to control retroposon transcript abundance (Djikeng et al 2001).…”