Nonsense-Mediated Decay in Trypanosomes Figure 5. Role of the 59-UTR length and translation. (A) Effect of the 59-UTR length on the CAT/NPT ratio. Stars indicate where paired values are different at the P = 0.05 level or below (Students t-test, two-tailed, unpaired). The sizes of the mRNAs were verified by Northern blotting (not shown). Details as in Figure 3. (B) Effect of the 59-UTR length on the amount of CAT protein. The amount of CAT protein was measured relative to aldolase (loading control). Results are the average of duplicate measurements, variation was very low. (C) Effect of the translation inhibition length on the CAT/ NPT ratio. The double ovals in the 59-UTRs represent hairpins which completely inhibit translation. Other details are as for Figure 4A. doi:10.1371/journal.pone.0025112.g005 Tethering of UPF1 to a 39-UTR can decrease mRNA abundance To find out whether UPF1 was capable of affecting mRNA levels at all, we used a tethering approach. As reporter, we used the CAT-ACT mRNA, with either 6 copies of the B box sequence, or two copies of MS2 stem-loop downstream of the stop codon (Figure 6A). Stable clonal cell lines were obtained that expressed the reporter mRNAs constitutively from the T7 promoter. These reporter lines were transfected with the plasmids giving inducible expression of lambdaN-tagged protein constructs: lambdaN- UPF1-flag (UPF1 with a C-terminal FLAG tag), lambdaN-GFP- TAP (Green fluorescent protein with a TAP tag) or UPF1-flag without the lambdaN (Figure 6B). A representative set of results obtained with the CAT-ACT 39UTR mRNA is shown in Figure 6C and quantitation for 3 biological replicates in Figure 6D. A reproducible decrease of the reporter mRNA was seen upon induction of lambdaN-UPF1-FLAG in the CAT-B-ACT cell line (Figure 6C&D, lanes 5,6). This decrease was dependent on the RNA-protein interaction because expression of lambdaN-UPF1- FLAG (Figure 6C&D, lanes 9,10) did not decrease the am- ount of CAT-MS2-ACT mRNA, Expression of UPF1-FLAG (Figure 6C&D, lanes 7,8) and tethering of lambdaN-GFP-TAP (Figure 6C&D, lanes 3,4) also did not decrease CAT-B-ACT mRNA. Results suggested that the UPF1 tethering was indeed affecting mRNA degradation (see Figure S4). Tethering of lambdaN-UPF1-FLAG to a target mRNA with a translation- inhibitory hairpin also caused a decrease in target mRNA (Figure 6E, lanes 5–8). Preliminary results (not shown) indicated that tethering of mutant upf1-1 also caused degradation. Overall, this would imply that TbUPF1 may be capable of recruiting the degradation machinery directly via protein-protein interactions. Effects of ORF and UTR lengths across the transcriptome To find out whether depletion of TbUPF1 had any effect on the steady-state transcriptome, we did microarray experiments using procyclic trypanosomes with TbUPF1 RNAi (full-length RNAi construct). No changes above two-fold were found, so the results have not been submitted to a database. Since a few probes showed up-regulation of 1.3–1.6-fold, we chose one - Tb927.10.12900 (up 1.54-fold) - for further analysis. Northern blotting revealed two bands:, one was the expected monocistronic RNA of 0.9 kb (increased 1.3-fold by UPF1 RNAi) and another of 4.5 kb that was PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25112