Figure 4

Sequence, structure and validation of predicted motifs. Probability matrix indicating the relative frequency of each residue at each position within the motif are shown, as well as secondary structures and structural alignment of five representative examples of motifs in specific transcripts. The alignment is represented by left and right balanced parenthesis indicating the pair of nucleotides that match by Watson-Crick complement or GU wobble pair. A) Best RNA motif identified for Tc UBP1 (UBP1m). B) Best RNA motif for Tc RBP3 (RBP3m). C) Scheme of the transcripts prepared for in vitro assays. PGEM-T was used as a control transcript because it lacks the motifs under analysis. Fragments of ZFP1 and RpS5 mRNAs bearing UBP1m and RBP3m, respectively, were inserted into PGEM-T. Dotted rectangle, location of motifs within each transcript. D) Biotin pull-down, GST, GST-Tc UBP1 and/or GST-Tc RBP3 (25 nM) were incubated with biotinylated transcripts (a) pGEM-T ZFP1m(+), (b) pGEM-T, (c) pGEM-T RpS5m(+). RNA-protein complexes were recovered using streptavidin-conjugated beads and detected by Western blotting. * degradation products of recombinant GST-Tc UBP1.