Extended Data Fig. 2: IsletMICs correspond to the subset of neuronal microexons with high sensitivity to SRRM3.
From: Pancreatic microexons regulate islet function and glucose homeostasis
(A, B) mRNA expression of the neural microexons regulator Srrm4 and of its paralog Srrm3 in neural tissues, pancreatic islets, exocrine pancreas and other rat (A) and mouse (B) tissues (data from VastDB, n = 41 in rat and n = 30 in mouse, biologically independent samples). Data are shown as box-and-whisker plot, in which the lower and upper bounds of the box represent the upper and lower quartiles, the centre line represents the median, and whiskers the 10th and 90th percentile. (C–E) siRNA-mediated knockdown (KD) of Srrm3 in INS-1E rat beta cell line. (C) Srrm3 mRNA levels measured by qPCR following 48 h transfection with Srrm3 (siSrrm3) or control (siCTL) siRNAs, normalized to Gapdh (n = 5 independent experiments). Data is shown as mean ± s.e.m. P values was obtained from Student’s two-tailed unpaired t-test. (D) RT-PCR assays for selected microexons in control and Srrm3 KD cells. The positions of inclusion/skipping isoforms and the percentage of microexon inclusion are indicated for two biological replicates. (E) Global impact of Srrm3 KD on exon inclusion levels estimated by PSI values from RNA-seq data. Differentially included exons in Srrm3 KD vs control are shown in orange (microexons, length [le] ≤ 27 nt), light blue (short exons, 27 < le ≤ 51 nt) and dark blue (cassette exons, le > 51 nt). The pie chart shows the number of misregulated exons (|∆PSI| > 15) according to their size range. Lower panel shows ΔPSI cumulative proportions for microexons, short exons and alternative cassette exons. P value was obtained from two-sided Wilcoxon test comparing the distributions of microexons and cassette exons of length > 51 nt. PSI values are the mean of three independent experiments. (F) Overlap between Srrm3-regulated microexons in rat INS-1E cells and rat IsletMICs. Only microexons with sufficient read coverage in both comparisons are shown. (G) Overlap between SRRM3-regulated microexons in rat INS-1E (R) and human EndoC-βH1 (H). Microexons presenting no ortholog (‘no orth’) or with no sufficient read coverage (‘no cov’) in the other species are indicated. (H) SRRM3 overexpression in HeLa cells at three different levels reveals different sensitivities for IsletMICs and neuronal-only MICs (NeuralMICs). Exon inclusion levels were quantified from RNA-seq and differences in inclusion between control and SRRM3-overexpressing cells are shown for IsletMICs and NeuralMICs (n = 1 experiment). Data are shown as box-and-whisker plot, in which the lower and upper bounds of the box represent the upper and lower quartiles, the centre line represents the median, whiskers the 1.5x interquartile range, and points the outliers.
