DRBD18 RNA binding and effects on the transcriptome. (A) Tagged DRBD18 was pulled down from bloodstream-form extracts, then associated (bound) RNAs were released using TEV protease. RNA that did not bind to the affinity beads served as the negative control (“unbound”). The average bound/unbound ratio for three biological replicates is on the y-axis and the annotated 3′-UTR length on the x-axis, both on a log scale. Each spot represents one open reading frame, with just one representative of repeated genes. The orange spots are genes with at least 10 reads in all samples and with at least twofold enrichment in all three comparisons. (B) Annotated mRNA lengths and 3′-UTR lengths for bound mRNAs (bloodstream forms, at least twofold enrichment in all three comparisons) and for mRNAs with, on average, less than onefold enrichment are plotted. The box plot shows the median and the box includes the interquartile range. Whiskers are up to 1.5-fold the inter-quartile ranges, and spots are outliers. (C) The 3′-UTRs of the 163 bound mRNAs (bloodstream forms) were compared with those of a length-matched set of mRNAs with less than onefold enrichment using MEME, using the discriminative mode and the more stringent differential enrichment modes. To the right are the numbers of bound mRNAs that contained the motif, and the probabilities from the program. (D) The log2 fold change for each coding region was subtracted from the log2 fold change of the corresponding 3′-UTR. If the result was less than −0.584 (1.5-fold excess of coding region reads) the mRNA was identified as having potential loss of 3′-UTR reads due to alternative polyadenylation. The numbers of genes for all three analyzed conditions are shown to scale: bloodstream-form whole-cell (WC) RNA, rRNA-depleted; procyclic-form whole-cell RNA, poly(A)+; and procyclic-form cytoplasmic RNA (Cyt), poly(A)+. Results were calculated for the “unique” gene set. (E) Effect of RNAi on the coding region/3′-UTR read ratio, plotted against the average DRBD18 RNA binding, in bloodstream forms. Results for unique coding regions and unique representatives of multigene families are shown. The orange dots are results for mRNAs that showed loss of 3′-UTR reads, as defined in D. (F) DRBD18 binding of mRNAs that showed loss of 3′-UTR reads relative to coding region reads, compared with binding of mRNAs that showed no (or less) 3′-UTR read loss. The Student t-test result is indicated. Results are for bloodstream forms. (G) Ratio of CDS reads in the cytoplasm (Cyt) relative to reads from the whole cell (WC), for procyclic forms. The Cyt/WC ratio with normal DRBD18 levels was subtracted from the ratio after RNAi. Positive results suggest an increase in the proportion of mRNA that was exported after RNAi. Results for the subset of mRNAs that showed loss of 3′-UTR reads relative to CDS reads are compared with the results for all other mRNAs. The Student t-test result is indicated.
