Nonsense-Mediated Decay in Trypanosomes Figure 4. Effect of the 39-UTR length and PABP on CAT mRNA abundance. (A) Effect of the 39-UTR length on the CAT/NPT ratio. The cartoons on the left illustrate the CAT mRNAs investigated. gfpNS: GFP coding region with no start codons. Details as in Figure 3. The sizes of the mRNAs were verified by Northern blotting (not shown). (B) Northern blot showing the sizes of selected reporter mRNAs. (C) Effect of UPF1 RNAi on the steady-state level of the 39++WT mRNA. Results are mean and standard deviation of 3 independent experiments. (D) Relationship between 39-UTR length (or termination codon – PABP distance) and mRNA abundance using data from (A) and (E). Results are mean 6 standard deviation. (E) Tethering of PABP1 and PABP2. Six copies of BoxB were inserted either between the EPTC CAT cassette and the 39-UTR in the EPTC 243 construct (Figure 3B), or after the AUG-less GFP coding sequence in the 39-UTR of the 39++WT construct (Figure 4A). Cell lines containing the various CAT mRNAs (as indicated to the left of the bar graph) were also transfected with tetracycline inducible plasmids expressing proteins bearing an N-terminal lambda-N peptide, and a C-terminal myc tag. Proteins were GFP, PABP1 (Tb09.211.0930) or PABP2 (Tb09.211.2150), shown to the left of the mRNA cartoons. Expression of the tagged proteins was induced for 48 h using 100 ng/ml tetracycline. The Western panels on the right, which were probed with antibody to the myc tag and to aldolase (control) come from a single experiment and exposed film. The amounts of CAT mRNA relative to NEO, measured by qPCR, are shown as mean and standard deviation for three independent experiments. doi:10.1371/journal.pone.0025112.g004 PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25112