Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei
A. Various fragments of RBP10 were inducibly expressed with an N-terminal lambdaN peptide and a C-terminal myc tag, in a cell line expressing a CAT reporter mRNA with 5 boxB sequences in the 3'-UTR. Cartoons showing the fragments are on the left. The numbers above the full-length protein are amino acid residues. The effects on the amounts CAT mRNA and CAT protein were measured by Northern blot and enzyme assay, respectively. Levels are expressed as mean ± standard deviation for at least 3 replicates, relative to a line with no lambdaN peptide protein (top bars). B. Bloodstream-form trypanosomes expressing the reporter in (A), and with tetracycline-inducible expression of lamdaN-RBP10-F3, were used. The reporter with or without tethered protein is shown at the top. Lysates from cells grown without (left) or with (right) tetracycline were subjected to sucrose gradient centrifugation. The panel below the cartoons show the absorption profiles at 254 nm as the fractions were collected. The migration of CAT and beta-tubulin (TUB) mRNAs on the gradient was detected by Northern blotting; representative blots are shown. The graphs show Northern signals, expressed as the percentage of the total signal, with arithmetic mean and standard deviation for three independent biological replicates.