The inner neurons, marked with yellow arrows, express almost exclusively GFP, an indication that the exon is skipped

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The inner neurons, marked with yellow arrows, express almost exclusively GFP, an indication that the exon is skipped. of the microexons marked with a red box Saikosaponin C on the left of the heat map are specifically included in retinal transcripts.(TIF) pgen.1006256.s001.tif (3.1M) GUID:?6DE00BEE-07BB-4A31-9556-FCA367FB63E6 S2 Fig: RT-PCR analysis of the inclusion levels of exons differentially spliced in wild type retina, Aipl1(-/-) retina, Nrl(-/-) retina and flow sorted rod photoreceptors. (A) Forward (FSC) vs Side scatter (SSC) scatter plots of dissociated retina from wild type and Aipl1(-/-) mice. The rod photoreceptor population is circled in red on the plot of the wild type retina cells. The rod population is absent from the Aipl1 knockout retina. (B) Quantitative RT-PCR analysis of the expression of photoreceptor, inner neuron and glial markers in the flow sorted rod photoreceptor cells, wild type retina and retina from Aipl1(-/-) mice. The expression levels of all marker genes are normalized to the levels in wild type retina. High levels of the rod transducin (Gnat1) are readily detectable in the flow sorted rod cell population, while the levels of inner neuron and glial cell markers were at or below the assay detection limits. (C) RT-PCR analysis of alternative splicing in wild type retina, Aipl1(-/-) retina, Nrl(-/-) retina and flow sorted rod photoreceptors. The bands corresponding to the exon skipped and exon included mRNA isoforms are labeled with + and -, respectively. The relative exon inclusion and standard error of three independent replicates are shown below each lane. (D) The difference in exon inclusion between rod photoreceptor cells and whole retina inversely correlates with the expression level of the gene in photoreceptors, approximated by the fold change in the expression between wild type and Aipl1 retina. The exon inclusion and gene expression levels were determined by RT-PCR and RNA-Seq, respectively. The inverse correlation is due to the mixed cell type composition of the whole retina and illustrates a limitation in our approach that may prevent the reliable discovery of photoreceptor specific splicing variants of genes with relatively low expression levels in photoreceptors compared to inner neurons.(TIF) pgen.1006256.s002.tif (3.3M) GUID:?CFF4422B-14D4-4465-A6A8-E46DDE14858C S3 Fig: Enrichment of MSI, PTBP and NOVA binding site motifs in clusters adjacent to exons upregulated in photoreceptors. Saikosaponin C Clusters with minimum size of 2, 3, 4 or 5 5 motifs were tested for each protein. The spacing between the motifs in a cluster was varied from 0 to 30nt (Xaxis). Enrichment upstream or downstream of the exons is plotted with circles and triangles, respectively. Statistically enriched clusters are represented by filled markers using red or blue colors for positions upstream or downstream of the exon, respectively.(TIF) pgen.1006256.s003.tif (3.5M) GUID:?76A30854-AB90-40F4-A686-BA67F0440819 S4 Fig: Enrichment of MSI, PTBP and NOVA binding site motifs in clusters adjacent to exons downregulated in photoreceptors. Clusters with minimum size of 2, 3, 4 or 5 5 motifs were tested for each protein. The spacing between the motifs in a cluster was varied from 0 to 30nt (Xaxis). Enrichment upstream or downstream of the exons is plotted with circles and triangles, respectively. Statistically enriched Saikosaponin C clusters are represented by filled markers using red or blue colors for positions upstream or downstream of the exon, respectively.(TIF) pgen.1006256.s004.tif (3.4M) GUID:?8A1E87AE-CF35-40FF-99EE-8B199BFCDE19 S5 Fig: Khdrbs3 is targeted by micro-RNAs from the mir-96/182/183 cluster. (A) Predicted binding sites for retinal micro-RNAs in the 3′-UTR of Khdrb3. PI4KA Binding sites conserved between mouse and human are shown in bold typeface. (B) Alignment of the retina specific micro-RNAs to the predicted binding sites. Saikosaponin C Seed sequences conserved between Saikosaponin C mouse and human are underlined. Each alignment is accompanied with mirSVR score representing the predicted efficiency of the target site (lower score means higher efficiency) and PhastCons sequence conservation score [76].(TIF) pgen.1006256.s005.tif (1.4M) GUID:?8F3C315F-E0F3-4AA5-95BA-718792971859 S6 Fig: Subcellular.