Stau1 regulates splicing of <i>INSR</i> exon 11 through an interaction with Alu elements in intron 10.

<p>(A) The genomic DNA sequence of the human INSR (NG_008852.1) was used to assess the Alu elements located in intron 10. Introns are not to scale, and this is indicated in intron 11 (//). The IR-minigene constructs used in this study are shown here. Previously deleted segments of genomic DNA determined not to influence exon 11 splicing are indicated in intron 11 (Δ symbol), and black dotted lines represent the deleted segment containing the three Alu elements. (B) HeLa cells were transiently transfected with Stau1-HA plasmid and either the WT or ΔAlus IR-minigene. Immunoprecipitation (IP) of Stau1-HA protein was carried out using HA-specific antibodies under RNase-free conditions. Western blot using HA-antibodies show equal amounts of Stau1-HA protein was immunoprecipitated in each condition. RNA was collected and DNase-treated prior to cDNA synthesis. RT-PCR was performed using <i>GAPDH</i> specific primers to demonstrate a lack of non-specific binding of RNA to the beads used for immunoprecipitation. Equal amounts of transfected minigenes were confirmed by performing RT-PCR on the cDNA synthesized from the 10% inputs lysates with primers specific to amplify a portion of the plasmid vector (pSG5) and the IR-minigene (Exon 10), corresponds to the pSG5-E10 labelled band. RT-qPCR was carried out using primers specific to an 115 bp region of intron 10 of the IR-minigene to determine the amount of IR-minigene RNA bound to immunoprecipitated Stau1-HA. Bar graphs show an average of four independent RIP experiments. (C-D) HeLa cells were transiently transfected with a CTRL, Stau1-HA plasmid or shStau1 and either the WT or ΔAlus IR-minigene. IR-minigene splicing was determined by RT-PCR. The average of ≥3 independent experiments was used. Error bars represent SEM * = p < 0.05, ** = p < 0.01, *** = p < 0.001.