Canonical as well as alternate binding motifs are enriched. (A,B). Most FBF peaks include the FBF binding element (FBE), and the occurrence of the FBE is proportional to peak height (FBE = UGUNNNAU, where N is any ribonucleotide). FBEs with a −1C or −2C, which confer higher affinity to the binding site, are enriched with opposite preference in FBF-1 and FBF-2. FBEs with an upstream cytosine at increasing distance (−3C and −4C) are less enriched. (C) The most significant motifs identified by MEME analysis of FBF peaks, after separating peaks with an FBE from peaks without an FBE. MEME analyses of peaks without an FBE reveal a shorter FBE-like 7-mer sequence. Thirty-one percent of all FBF-1 peaks and 33% of all FBF-2 peaks had a 7-mer. (D) Searching for a subset of alternate motifs exclusive of the canonical FBE (which is shown in line 0 in the table) in all peaks reveals significant enrichment (P < 0.01) relative to randomly shuffled peak sequences. Lines 1, 2, and 3 represent half-mer sequences based on the UGU trinucleotide characteristic of all PUF protein binding sites. Lines 4, 5, 6, and 7 are motifs identified in an in vitro selection study.
