RNA interference, growth and differentiation appear normal in African trypanosomes lacking Tudor staphylococcal nuclease

Sam Alsford,Louise E Kemp,Taemi Kawahara,David Horn

doi:10.1016/j.molbiopara.2010.06.006

Sam Alsford, Louise E Kemp + Show 2 more

Open Access

https://doi.org/10.1016/j.molbiopara.2010.06.006

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Abstract

Ribonucleases play important roles in the RNA interference (RNAi) pathway. The Dicer endonuclease converts double-stranded (ds)RNA into small interfering (si)RNA and the Slicer endonuclease, as a component of the RNA induced silencing complex (RISC), cleaves mRNA. Tudor staphylococcal nuclease (Tudor-SN) is another component of RISC in humans, flies and nematodes and is therefore implicated in the RNAi pathway. Here, we explore the potential role of African trypanosome Tudor-SN in RNAi. First, we assembled tudor-sn null mutants and showed that the gene is dispensable for normal growth and for differentiation. Next, we developed an inducible RNAi reporter system and demonstrated that Tudor-SN is dispensable for RNAi. The kinetics of mRNA knock-down, protein knock-down and protein recovery following inactivation of dsRNA expression are all unperturbed in the absence of Tudor-SN. We conclude that if this nuclease plays a role in the destruction or processing of dsRNA, mRNA or siRNA in the RNAi pathway, it is likely a minor one.

Highlights

Ribonucleases play important roles in the RNA interference (RNAi) pathway
Once siRNA is loaded into the RNA induced silencing complex (RISC), other RNases such as the argonaute protein, RDE-1 [5] and C3PO, a complex of Translin and Trax [6], promote RISC activation by removing the passenger strand from the siRNA duplex
RNAi does not operate in all trypanosomatids, but in African trypanosomes, two Dicer nucleases, DCL1 and DCL2 [9], and a Slicer nuclease, AGO1 [10,11], function in RNAi; a role in transposon silencing has been demonstrated [12] and RNAi is widely used as a functional genomics tool [13,14]