Fig. 2.

Predicted secondary structures of the mutant CD22 pre-mRNA sequences in infant BPL cells. Prediction of secondary structure of the positive strand of the RNA molecule, to assess the potential for RNA binding proteins to target motifs, was achieved using the minimum free energy (MFE) calculations for sequences obtained from intronic regions between exons 12 and 13 (RNAfold, provided by the Vienna RNA package). (A.1) The folded structure for the wild-type sequence with the target motifs for the splicing factors hnRNP-L, PTB, and PCBP. The positions of the misalignments caused by genomic mutations for each patient are indicated by the arrow symbols [alignments performed using the Clust W algorithm (Bioedit Sequence alignment editor)]. The misalignments occur in the RNA helix portion of the wild-type folded structure and lead to marked changes in the predicted secondary structure for three of the patients with complete sequences in the intronic region (A2; PT1, PT5, PT6). (A.2) Whereas the wild-type secondary structure contained 11 hairpin loops, 2 bulges, 6 multibranched loops, and 8 internal loops in the RNA helix, there were 9 hairpin loops, 4 bulges, 5 multibranched loops, and 11 internal loops in PT1 secondary structure; 10 hairpin loops, 3 bulges, 6 multibranched loops, and 10 internal loops in PT5 secondary structure; and 7 hairpin loops, 3 bulges, 5 multibranched loops, and 14 internal loops in PT6 structure. (B.1 and B.2) The CACA binding motif for hnRNP-L appeared at a base of a multibranched loop comprised of two hairpin loops in wild-type and PT5 pre-mRNA, while this motif was sequestered between an internal loop and a multibranched loop through formation of a double strand between CAC and GUG complementary pairs in pre-mRNA from PT1 and PT6. A second loop structure with an ACAC binding motif showed open access in a hairpin loop structure from wild-type and PT5 pre-mRNA and apparent potential for steric hindrance adjacent to a region with an internal loop and a bulge in pre-mRNA from PT1 and PT6. (C.1 and C.2) A PTB-binding site UCU showed two bases in a hairpin loop structure of wild-type pre-mRNA. Notably, in PT1 and PT6 all three bases appear within the hairpin loop at the end of a stem with 10 base pairs making this motif more accessible for PTB binding. An alternative PTP-binding site (viz.: CCU) formed a junction between a multibranched loop and a stem in wild-type pre-mRNA and PT5 pre-mRNA, but in the other two patients the GGG is double-stranded making this motif inaccessible to protein binding. (D.1 and D.2) There were two binding sites for PCBP that exhibited variation in the binding site accessibility and surrounding structural conformations for the wild-type and patient sequences. In one motif, the multibranched portion of the double hairpin loop structure contained a triple C site and the junction with the helix contained the quadruple C site with low base-pair binding probabilities in the wild type, whereas the patient secondary structures showed complex folding patterns that resulted in close proximities of adjacent stem-loop structures that could potentially hinder PCBP binding events. The second binding site for PCBP was found in a bulge portion that also contained a single stem-loop structure. This site contained 5 Cs in the wild-type and PT5 RNA sequences, whereas in PT1 and PT6 this bulge region collapsed making the motif inaccessible to binding.