Comparative genomics reveals two novel RNAi factors in Trypanosoma brucei and provides insight into the core machinery

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.

Figure 1. Characteristics of TbRIF4.

Schematic representation of TbRIF4 (the drawing is not to scale) and alignment of the three motifs characteristic of 3′-5′ exonucleases of the DEDDh superfamily from E. coli DnaQ , N. crassa QIP , and T. brucei RIF4. Asterisks and bold characters indicate conserved residues.

Figure 2. Analysis of TbRIF4- and TbRIF5-mutant cell lines.

(A) Response of rif4^−/− cells and cells expressing TbRIF4 catalytic mutants or HsAGO2 to transfection of α-tubulin dsRNA. Cells were electroporated with different amounts (in micrograms) of α-tubulin dsRNA, as indicated above each lane or with 5 µg poly(I-C) (first lane in each panel set), and total RNA was prepared 2 h after electroporation and analyzed by Northern hybridization with an α-tubulin-specific probe (α-tubulin panels). α-tubulin mRNA hybridization was quantitated by PhosphorImager analysis, normalized to the load control and expressed as % mRNA remaining, setting as 100% the amount of α-tubulin mRNA present in the samples that received poly(I-C). Load; hybridization to fumarate hydratase mRNA. (B) Steady-state levels of repeat-derived (CIR147) transcripts are increased in rif4^−/− cells but not rif5^−/− cells. Total RNA isolated from various cell lines, as indicated above each lane, was analyzed by Northern hybridization with a CIR147-specific probe. Load; hybridization to α-tubulin mRNA. Filled squares indicate the positions of the large ribosomal RNAs, as determined by methylene blue staining of the membranes. (C) Semi-quantitative RT-PCR analysis of CIR147 transcript levels. cDNA derived from various cell lines (as indicated above each lane; lanes 1–10) was used as a template for 22 cycles of PCR using oligonucleotides that amplify the CIR147 tandem repeat transcripts, producing a ladder of fragments (top two panels), or a portion of histone H4 (bottom two panels). Dilutions of genomic DNA were used as a positive control and an indicator for non-saturated PCR (lanes 11–13); mock cDNA synthesis without reverse transcriptase (second and fourth panels, -RT) served as a negative control. (D) Cells were visualized using differential interference microscopy (DIC), GFP imaging, and Hoechst (Nucleus and Kinetoplast are indicated by N and K, respectively); GFP and Hoechst images were merged (merge).

Figure 3. Analysis of siRNAs in rif4^−/− and rif5^−/− backgrounds.

(A) Steady-state siRNA levels in rif4^−/− and rif5^−/− cells. Low-molecular weight RNAs were separated by denaturing gel electrophoresis and analyzed by Northern hybridization with an ingi-, SLACS- or CIR147-specific probe, as indicated next to each panel. Load; hybridization to 5S rRNA. 26 nt, DNA marker. (B) Dicing is unaffected in ago1^−/− and rif4^−/− T. brucei extracts, but shows an altered pattern in rif5^−/− extracts. Whole cell extracts from cell lines, as indicated above the lanes, or buffer alone (−) were incubated with a 83-nt, internally labelled dsRNA substrate. After incubation the products of digestion were separated on a denaturing polyacrylamide gel. Size marker positions are indicated. (C) Native gel analysis of siRNAs from rif4^−/− and rif5^−/− cells. Low molecular weight RNAs isolated from various cell lines, as indicated above each lane, were resolved on a native polyacrylamide gel without (−) or with (+) heating the samples to 100°C for 2 minutes prior to electrophoresis and analyzed by Northern blotting with a CIR147 probe. A radiolabelled synthetic RNA was included as a control (lanes 7 and 8). Load; hybridization to 5S rRNA.

Figure 4. siRNAs are not associated with TbAGO1 in rif4^−/− cells.

(A) Cytoplasmic extracts from cells expressing TAP-tagged TbAGO1 in a wild-type (lanes 1–4) or rif4^−/− (lanes 5–8) background were immunoprecipitated with IgG beads, and the indicated amounts of supernatant (S, lanes 1 and 5) and immunoprecipitated material (P, lanes 2–4 and 6–8) were analyzed by Western blotting with a polyclonal anti-TbAGO1 antibody (upper panel, TbAGO1). CIR147 siRNAs in the soluble and precipitated materials were revealed by Northern hybridization (middle panel, CIR147 siRNA). RNA loading was quantified by hybridization to 5S rRNA (bottom panel, 5S RNA). (B) TbRIF4 and TbAGO1 steady-state levels in various cell lines. Total protein extracts from 5×10⁶ cells from various cell lines, as indicated above each lane, were resolved by SDS-PAGE and Western blotted with a polyclonal anti-TbRIF4 antibody (top panel, TbRIF4) or a polyclonal anti-TbAGO1 antibody (middle panel, TbAGO1). Load; a cross-reacting band to the TbAGO1 antibody (bottom panel).

Figure 5. Recombinant TbRIF4 3′-5′ exonuclease activity in vitro.

(A) Titration of GST-RIF4 activity. 1 pmol of a 25 nt-long, 5′-end labelled synthetic dsRNA was incubated with 0.01–30 pmol GST-RIF4 (lanes 4–11; molar ratios from 10∶1 to 1∶30) or 30 pmol GST-TbRIF4 carrying mutation H472A (lane 12). Lane 3, input. AH, alkaline hydrolysate ladder. M, marker oligonucleotides. Samples were separated on a denaturing 20% polyacrylamide gel. (B) Action of GST-TbRIF4 on a RNA substrate blocked at the 3′ end of one strand. 1 pmol of a 25 nt-long, 5′-end labelled synthetic dsRNA with a dideoxycytosine residue at the 3′ end of the labelled strand was incubated with 0.03–30 pmol GST-TbRIF4 (lanes 3–6), or buffer alone (lanes 1 and 2). Samples were separated on a non-denaturing 16% polyacrylamide gel. As a control for the separation of double-stranded and single-stranded species, the substrate incubated with buffer alone was resolved on the same gel without (lane 1, native) or with (lane 2, denat.) heating to 100°C for 2 minutes prior to electrophoresis.

Figure 6. Interaction between TbRIF4, TbAGO1 and siRNAs.

(A) Cytoplasmic extracts from rif4^−/− cells expressing wt TbRIF4-GFP (lanes 1–3) or mutant H472A TbRIF4-GFP (lanes 4–6) were subjected to immunoprecipitation with anti-GFP antibody. 0.1% of the supernatant (S, lanes 1 and 4), 0.1% (lanes 2 and 5) and 0.4% (lanes 3 and 6) of the immunoprecipitated material were analyzed by Western blotting with a polyclonal anti-RIF4 antibody (top panel) or a polyclonal anti-TbAGO1 antibody (bottom panel). The efficiency of immunoprecipitation is shown in the antibody titration experiment in Figure S7. (B) The presence of CIR147 siRNAs in the various samples as described in panel (A) was analyzed by Northern blot hybridization with a CIR147 probe. Load; hybridization to 5S rRNA. (C) Immunoprecipitated HsAGO2 is associated with duplex and single-stranded siRNAs. Cytoplasmic extracts from cell lines expressing HA-FLAG-HsAGO2 in an ago1^−/−:rif4^−/− genetic background, were immunoprecipitated with anti-FLAG antibodies. RNA associated with HsAGO2 was separated on a native polyacrylamide gel and subjected to Northern hybridization with a CIR147 probe (upper panel). Total RNA from rif4^−/− trypanosomes was used as a control for the position of duplex siRNAs (T, lane 1). Load; hybridization to 5S rRNA (middle panel). Equal amounts of the supernatant (S, lane 2) and immunoprecipitated material (P, lane 3) were analyzed by Western blotting with anti-HA antibody (HsAGO2 panel).