FIGURE 1.

MBNL1 autoregulated splicing. A, two MBNL1 isoforms are depicted with exons as gray boxes. The top isoform contains exon 5. The MBNL1 pre-mRNA contains an ultraconserved region spanning exon 5 through intron 4 shown by a PhastCons plot of placental mammal conservation. RNA structural elements upstream and within exon 5 were predicted by EvoFold (19) and are represented by black boxes with arrows. This figure was edited from the UCSC Genome Browser (GRCh37/hg19 assembly) (44). B, sequence alignment between MBNL1 and MBNL2 shows the 3′-end of intron 4, exon 5, and the highly conserved region of intron 5. Intronic sequence is lowercase, and exon 5 is capitalized. Possible MBNL1/MBNL2 pre-mRNA distant branch points and PY tracts are in bold, and YGCY (MBNL1 binding sites) motifs are highlighted in black. The underlined sequence is the ultraconserved element described by Bejerano et al. (16). Intron 4 of MBNL1 contains a weak AAG 3′-ss, and intron 5 of MBNL1 contains a weak GTACTA 5′-ss. C, schematic of the wild type MBNL1 minigene containing exon 4, intron 4, exon 5, intron 5, and exon 6. D, in vivo splicing of the MBNL1 minigene showing that exon 5 exclusion of the wild type minigene is MBNL1-dependent. Lane 1 shows the wild type splicing of the MBNL1 minigene, lane 2 shows the splicing results from co-transfection of the MBNL1 minigene and CUG₉₆₀ repeat plasmids, and lane 3 shows the results from the co-transfection of the MBNL1 minigene and MBNL-eGFP plasmids. nts, nucleotides.