Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei
A) Abundances of mRNAs that were at least 3x enriched in both RBP10-bound fraction, relative to those that were less than 1x enriched. B) The 3'-UTRs of 188 mRNAs that were at least 3x enriched in both RBP10-bound samples (Supplementary S2 Table) were compared with those of mRNAs that did not bind (<0.7x enrichment in both experiments) using DREME. Only 3'-UTRs annotated in tritrypDB were used (S1 and S2 Text) and some were not mapped with sufficient confidence to be included. The best-scoring motif found is shown in the inset. The graph shows numbers of UA(U)6 motifs in 255 manually annotated bound 3'-UTRs (S2 Table). One mRNA each had 7, 8 and 9 motifs. The numbers of motifs shown here are sometimes higher than with automatically annotated 3'-UTRs from the genome database, because some of the automatic 3'-UTRs are truncated. C) Effect of the UA(U)6 motif on RBP10 binding: median ± 25th percentiles, with 95% confidence limits and outliers. Database 3'-UTRs were analyzed. Since these are often truncated, and are missing for 30% of genes, the numbers of motifs are under-estimated. Significant differences (Student t-test) are shown. D) Reporters used in (E) and (F) . The red bars are UAUUUUUU elements, which are highlighted in the 26mer sequence. E) RBP10 binding to the EP 3'-UTR requires the 26mer. Cells were UV-irradiated, and RBP10 was pulled down with anti-RBP10 (upper panel, Western blot). RNAs were detected by reverse transcription and PCR using gene-specific primers (lower panel). If reverse transcriptase was omitted, no PCR band was obtained. F) rbp10 RNAi specifically affects expression of a reporter containing the 26mer. CAT activity was measured 17h and 24h after RNAi induction.