HITS-CLIP data, RNA read assignments, and quantification. (A) Illumina CLIP sequencing data and RNA tag count data. Pie charts depict the relative abundance of the RNA targets, which are derived from the RNA tag count data. Coding sequences are defined as the annotated open reading frames of mRNAs in version 4 of the T. brucei brucei 927 genome at TriTrypDB and GeneDB (tritrypdb.org/tritrypdb and www.genedb.org). Intercoding sequences contain 5′ and 3′ UTRs, as well as sequences that may contain primary transcripts. rRNA/tRNA, anti-sense, and other RNAs are as defined in the database referenced above. Reads with poor quality and sequence ambiguity, as well as reads generated by PCR amplification, were eliminated prior to mapping. (B) RBP42–mRNA interactions and DRBD3–mRNA interactions (control protein–RNA interactions) within specific genes in the T. brucei genome. (Profile 1) RBP42-bound sequences within the coding region of succinyl-CoA synthetase (α-subunit shown here; Tb927.3.2230). Two upstream genes (Tb927.3.2250 and Tb927.2240) and one downstream gene (Tb927.2220) that are not bound by RBP42 reveal binding specificity, as does the absence of DRBD3 binding to all four genes shown. (Profile 2) RBP42-bound sequences within the CDS and DRBD3-bound sequences within the 3′ UTR of Gim5B (Tb09.211.2740). The absence of the reciprocal binding, as well as the absence of binding to the flanking genes, indicates the binding specificities. (Profile 3) DRBD3-bound sequences within the (predicted) 3′ UTR region of MFS mRNA (major facilitator superfamily protein; Tb927.8.7740). DRBD3-targeting of the MFS mRNA 3′ UTR was initially discovered by Estevez (2008). (Binding specificity is revealed by the low binding of DRBD3 or RBP42 to the CDS of this gene, as well as the low binding of RBP42 to the 3′ UTR of this gene. The small amount of signal indicating DRBD3 interactions with the 3′ UTR of the upstream gene, Tb927.8.7730, was not investigated.) (Profile 4) DRBD3-bound sequences within the coding region of a hypothetical protein mRNA (Tb927.1.2140). Binding specificity is revealed by the absence of RBP42 binding to this gene, as well as the absence of DRBD3 and RBP42 binding to two upstream flanking genes (Tb927.1.2120 and Tb927.1.2130) and the downstream flanking gene (Tb927.1.2150). Scales, 0–1500 (Profile 1), 0–300 (Profile 2), 0–100 (Profile 3), and 0–600 (Profile 4). Green denotes RBP42-bound sequences, and orange denotes DRBD3-bound mRNA sequences.
