Mutation of two intronic nucleotide alters RNA structure and dynamics, inhibiting MBNL1 and RBFOX1 regulated splicing of the Insulin Receptor

Abstract

Alternative splicing (AS) of Exon 11 of the Insulin Receptor (INSR) is highly regulated and disrupted in several human disorders. To better understand INSR exon 11 AS regulation, splicing activity of an INSR exon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/574689v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e410org.highwire.dtl.DTLVardef@7deb00org.highwire.dtl.DTLVardef@cf69f9org.highwire.dtl.DTLVardef@13684e8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure:C_FLOATNO Model for INSR exon 11 splicing regulation through the UGCAUG motif. We propose that the UGCAUG motif, MBNL1, and RBFOX1 cooperatively regulate INSR exon 11 splicing. Mutating this UGCAUG motif is sufficient to alter RNA structural dynamics to disrupt this regulation. C_FIG