Experiments were done using using bloodstream-form Lister 427 strain T. brucei. Plasmids and oligonucleotides are listed in Supplementary dataset file 1. EC50 determinations and Northern blotting were done exactly as described previously [17]. For the pull-down experiment, the cells were exposed to AN7973 at 10x EC50 for 30 min (15 min followed by 13 min centrifugation). The tagged protein was then purified from 1 × 109 cells (at ~ 1 × 106 cells/ml) exactly as described in [19]. Briefly, the protein was allowed to adhere to IgG magnetic beads. After washing, the tagged protein was released using His-tagged tobacco etch virus protease, which was then depleted using nickel-derivatized magnetic beads. We examined four replicates for CPSF73-TAP both with and without AN7973, and for GFP-TAP, one preparation with, and one without, AN7973. The methods for mass spectrometry were as previously described for the RNA-binding protein RBP10 [20]. The samples were run briefly on an SDS polyacrylamide gel and analyzed by mass spectrometry at the ZMBH Core facility. Statistical analysis was performed using Perseus version 1.6.15.0 [21].
Results and discussion: splicing inhibition
We first measured splicing inhibition. Preliminary measurements yielded a sub-nanomolar EC50 for AN11736, and an EC50 of 512nM for acoziborole. We also confirmed the observation [13] that the carboxylate metabolite of AN11736 (Fig. 1) was much less active than the parent compound: it had no detectable anti-trypanosomal activity at the concentrations tested. To detect splicing inhibition, RNA was collected at different time-points after treatment with 10x EC50, which was 6.3 nm for AN11736 (based on published values) and 5.12 μm for acoziborole. Levels of spliced total mRNA and β-tubulin mRNA were then evaluated using Northern blots exactly as previously described [17]. Methylene blue staining, which detects the stable (non-spliced) rRNAs served as the control (Fig. 2A, panel a). Spliced mRNAs were detected by probing the blot with a 39mer oligonucleotide complementary to the spliced leader (SL): this detects both processed mRNAs, and the spliced leader RNA (SLRNA) substrate for the trans splicing reaction. Treatment with acoziborole resulted in gradual reduction in spliced mRNAs (Fig. 2A, panel b). The level of SLRNA was probably unaffected (although it is difficult to quantify due to over-exposure), suggesting no substantial inhibition of RNA polymerase II transcription. The blot was then stripped and probed for the β-tubulin (TUB) mRNA. As previously observed using AN7973, partially-spliced mRNAs containing two or more tubulin open reading frames accumulated (Fig. 2A, panel c). After 4 h of drug exposure, there was a reduction in total mRNA and TUB mRNA. We speculate that at this point, even partial processing is no longer possible and unprocessed mRNA precursors are degraded in the nucleus. Similar results were obtained after treating the cells with AN11736, except that fewer tubulin precursors were detected (Fig. 2B). These findings confirm that AN11736 and acoziborole indeed inhibit mRNA processing.
Results and discussion: CPSF73-associated proteins
We had speculated that CPSF73 binding might stabilise the polyadenylation complex and its interaction with the splicing machinery. To test this, we compared the proteins that copurified with affinity-tagged CPSF73 with or without prior treatment with AN7973. We first integrated a sequence encoding a tandem affinity purification (TAP) tag downstream of, and in frame with, the CPSF73 open reading frame (Additional file 1). CPSF73 is an essential gene [15], so to check that the tagged protein was functional, we deleted the wild-type allele and monitored cell growth. In comparison to wildtype cells, the TAP-CPSF73 cells grew slightly slower (Fig. 3A). This might have been a consequence of copy-number reduction since cells with a single wild-type gene also grew at the same slightly slower rate (Fig. 3A). Also, we had replaced the 3’-untranslated region of the tagged allele, which could affect expression. We did not, however, assess CPSF73 protein levels.
Next, we evaluated the cells’ sensitivity to AN7973. For wild-type cells, the average EC50 from 3 independent experiments was 22.9 nM (Fig. 3B), agreeing with our previous results [17]. Surprisingly, the CPSF73-TAP cell lines were approximately five times more sensitive to AN7973 than the starting cell line, with an EC50 of 4.2nM (Fig. 3B). This might be due to a decreased amount of CPSF73, but this has not been verified. An effect of the tag cannot be ruled out.
Next, we determined the effect of AN7973 on the expression of CPSF73-TAP. Parasites were diluted to 1 × 105 cells/ml and grown for 24 h to final concentration of ~ 1 × 106 cells/ml. The culture was treated with 10x EC50 AN7973 for 6 h. Cells were collected for western blot analysis before adding the drug, and at various times thereafter. The amount of CPSF73-TAP was calculated relative to untreated cells (time point 0 h) and the ponceau red stain was used as a loading control. There was a 30% decrease in CPSF73-TAP protein after four hours (Fig. 3C). Previous in vivo [35 S]-methionine incorporation assays had showed a dramatic decrease in total protein synthesis only after 4 h of AN7973 exposure [17], but this was almost certainly secondary to loss of mRNA. The half-life of CPSF73 was measured, using pulse-chase and mass spectrometry, to be about 5.5 h [22]. Although the half-life of the CPSF73-TAP mRNA is unknown, the decrease in CPSF73-TAP protein after AN7973 treatment could have been due to loss of functional mRNA and therefore, loss of CPSF73 protein synthesis.
To detect effects of AN7973 on protein associations of CPSF73-TAP, cells were either untreated, or exposed to 10x EC50 AN7973 for 30 min. Four replicates each were examined. As an additional control, cells expressing GFP-TAP were used, with just two replicates since the composition of the polyadenylation complex is already well known [15]. Tagged proteins were purified and identified by mass spectrometry. Regardless of drug treatment, CPSF73-TAP copurified with members of the polyadenylation complex (Additional file 2): all the CPSF subunits (CPSF7160/100/30/60/73 and Fip1), CstF50, Simplekin and two proteins that co-purify with CPSF160 (Tb927.11.13860 and Tb927.8.4480) [15]. Spliceosome components were not co-purified. The only difference that we noticed after drug treatment was that ubiquitin was detected in three out of four replicates of drug treated samples while it was only detected in one replicate in the untreated cells. However, this difference was not statistically significant, and ubiquitin was also detected in one of the GFP replicates. These results show that AN7973 treatment for 30 min has no significant effect on proteins that co-purify with CPSF73.