(A) Time course of TbAGO1 mRNA disappearance after removal of tet. TbAGO1KO_c cells were washed twice in medium and then put back in culture in tet-minus medium for the times indicated above each lane. RNA was extracted and analyzed by Northern blot using a TbAGO1 coding-region probe. Note that two hybridizing bands are seen in lane 1; they are derived from utilization of two alternate 3′-splice sites present upstream of the TbAGO1 coding region. Control: the membrane was stripped and rehybridized with a probe detecting the mitochondrial large ribosomal RNA. (B) Decay of TbAGO1 mRNA in wild-type trypanosomes. Wild-type trypanosomes were incubated with the transcription inhibitor actinomycin D (AMD) as previously described (Shi et al. 2004b). RNA samples were prepared at the times indicated above each lane and analyzed by Northern blotting with an AGO1 coding-region probe. Control: hybridization to the 18S rRNA as a loading control. (C) Time course of the disappearance of TbAGO1 after tet removal. TbAGO1KO_c cells were washed and incubated in tet-free medium as described in the legend of A. Whole-cell extracts were fractionated on a 6% SDS-PAGE and processed for Western blotting with anti-AGO1 antibodies (Shi et al. 2004b). Control indicates a cross-reactive protein. (D) Endogenous TbAGO1 is a stable protein. A cell line expressing BB2-TAP-tagged TbAGO1 (Shi et al. 2004b) was metabolically labeled with ³⁵S-methionine as described in Materials and Methods. After a 2-h pulse, the label was chased with an excess of unlabeled methionine, and cytoplasmic extracts were prepared at the times indicated above each lane. TbAGO1 was then immunoprecipitated with anti-BB2 antibodies, the immunoprecipitates were separated by SDS-PAGE, and the radioactive bands were detected by autoradiography. The panel chase control shows a group of bands from the total protein samples run in parallel to show that the chase was effective. As a control for the efficacy of the immunoprecipitation, the membrane shown in the upper panel was analyzed by Western blotting with the anti-BB2 antibody, which detects the total amount of AGO1 protein present in the immunoprecipitates.

(A) Conditional KO of TbAGO1 results in up-regulation of large transcripts homologous to the Ingi retroposon transcripts. Total RNA was prepared from cells grown in the presence or absence of tet for the times indicated above each lane. Ingi transcripts were detected with an Ingi probe as described (Janzen et al. 2006). Control: hybridization to the calflagin mRNAs. (B) Conditional KO of TbAGO1 results in an inhibition of Ingi siRNA accumulation. TbAGO1KO_c cells were incubated in the presence (+) or absence (−) of tet for 24 h. Total RNA was prepared and enriched for small RNAs as described previously (Shi et al. 2004b). The small RNAs were separated on a denaturing 15% polyacrylamide gel, electroblotted onto a nylon membrane, and hybridized with a sense radiolabeled riboprobe representing approximately the first 1000 nt of the Ingi coding region. Control shows a group of cross-hybridizing bands. The position of a 26-nt DNA marker is shown by a bar on the side.

(A) Transfection of a morpholino antisense oligonucleotide inhibits accumulation of newly synthesized TbAGO1. Cells solely expressing BB2-TAP-tagged TbAGO1 were transfected with an AGO1 antisense morpholino oligonucleotide or with an oligonucleotide with the inverse sequence as a negative control (for details, see Materials and Methods). Four hours after transfection, the cells were metabolically labeled with ³⁵S-methionine for 2 or 4 h, and cytoplasmic extracts were prepared and immunoprecipitated with anti-BB2 antibodies. The immunoprecipitates were separated by SDS-PAGE and the radioactive bands detected by autoradiography (upper panel). The same membrane was reacted with anti-AGO1 antibodies to verify the efficiency of immunoprecipitation (lower panel). (B) Transfection with the AGO1 antisense morpholino oligonucleotide inhibits degradation of α-tubulin mRNA. A trypanosomes cell line conditionally expressing α-tubulin dsRNA, pLEWFAT (Shi et al. 2000), was used for this analysis. Cells were transfected with the AGO1 antisense or inverse morpholino oligonucleotide, or mock-transfected (no oligo), and grown for the periods of time indicated above each lane in medium containing 10 μg/mL tet, which induces maximal synthesis of α-tubulin dsRNA. At each time point, RNA was extracted and analyzed by Northern blotting with an α-tubulin coding-region probe or with a control probe for actin mRNA. The percent α-tubulin mRNA degradation was calculated as described in the legend to Figure 2. (B) The steady-state level of TbAGO1 does not significantly change upon transfection with morpholino oligonucleotides. pLEWFAT cells were transfected with the indicated morpholino oligonucleotides and grown in the presence of tet as described above. Samples were withdrawn at various intervals (indicated above each lane) and processed for Western blot analysis with anti-AGO1 antibodies. Control indicates a cross-reacting protein.

(A) Disappearance of AGO1 protein in response to decreasing tet concentrations. TbAGO1KO_c cells were grown in the absence of tet for 4 d, and then incubated for 24 h with different tet concentrations (lanes 1–5) or without tet (lane 6). Cytoplasmic extracts were analyzed by Western blotting with an anti-AGO1 antiserum. Tet concentrations: (lane 1) 10 μg/mL; (lane 2) 1.0 μg/mL; (lane 3) 0.1 μg/mL; (lane 4) 0.01 μg/mL; (lane 5) 0.001 μg/mL. (Cont) A cross-reactive protein. The amount of AGO1 was quantitated from the autoradiogram and is expressed as the percent of AGO1 expressed at the highest tet concentration. (ND) Not detectable. (B) The RNAi competency of TbAGO1KO_c cells decreased in response to incubation with decreasing amounts of tet. TbAGO1KO_c cells were grown using the same tet concentrations as described in A. After 24 h, the cells were electroporated with 2 μg of α-tubulin dsRNA and then returned to the medium for 2 h. Ten micrograms of total RNA was analyzed by Northern blotting using an α-tubulin coding-region probe. The hybridized membranes were analyzed by PhosphorImaging, and the percent α-tubulin mRNA degradation was calculated, after adjusting for loading differences, by taking the amount of α-tubulin mRNA present at time 0 as 100%.

(A) Inhibition of TbAGO1KO_c RNAi competency after 4 h of growth in the absence of tet. Cells were washed free of tet and incubated for 2 h without or with addition of tet. Next, cells were simultaneously transfected with different amounts of α-tubulin and PFR dsRNA as indicated above each lane, and total RNA was isolated 2 h later. Ten micrograms of RNA was analyzed by Northern blotting with the following probes: a portion of the α-tubulin coding region (upper panel); a portion of the PFR coding region (middle panel); a portion of the actin mRNA as a loading control. The percent mRNA degradation was calculated as described in the legend to Figure 2. (B) The steady-state amount of TbAGO1 does not change within 4 h from tet removal. Cells were washed free of tet and immediately lysed with SDS-PAGE (time 0) or grown for 2 h, transfected with dsRNA, and then lysed 2 h later (time 4 h). The presence of AGO1 was revealed by Western blotting with the anti-AGO1 antiserum. (Con) A cross-reacting band used as a loading control. (C) The steady-state level of Ingi transcripts does not change during 5 h of incubation in the absence of tet. Ten micrograms of total RNA extracted from cells immediately after tet removal (time 0) or after 2 or 5 h of incubation in the absence of tet was analyzed by Northern blotting with an Ingi coding-region probe. (Con) 18S ribosomal RNA as a loading control.