Abstract
The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3′-5′ exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.
Highlights
RNA interference (RNAi) was first described in 1998 and within a short period of time tremendously facilitated the analysis of gene function, especially in organisms where classical genetic approaches are not available
In order to identify the core RNAi genes we compared the genomes of RNAi-deficient (T. cruzi and L. major) and RNAiproficient (T. brucei, T. congolense and L. braziliensis) trypanosomatids and selected ORFs that are exclusively present in all the RNAipositive organisms, but absent from the RNAi-negative parasites
The comparative genomics analysis revealed a limited set of five genes: the previously characterized TbAGO1, TbDCL1 and TbDCL2, as well two additional candidates, TbRIF4 and TbRIF5
Summary
RNA interference (RNAi) was first described in 1998 and within a short period of time tremendously facilitated the analysis of gene function, especially in organisms where classical genetic approaches are not available. The very recently introduced RIT-Seq method (RNA Interference Target Sequencing) took advantage of the power of genome-wide RNAi screens and combined it with the strength and depth of next-generation Illumina sequencing [3]. This strategy provided the scientific community with a catalogue of genes whose knockdown is detrimental to the parasite under a variety of developmental conditions and is likely to find numerous applications in RNAi-positive parasites. The recent success of David Bartel’s group in reconstructing the RNAi pathway in S. cerevisiae [9] provides a proof of principle and informs us that it is realistic to try to achieve the same goal in T. cruzi or old world Leishmania sp
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
