Additional file 8: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

Sequence of all primers generated for this study, together with qPCR probes, amplification efficiencies, and primer/probe combinations used. Exon content of amplicons used for Northern analyses is also shown. For additional assays, see [7]. (XLSX 12 kb

Additional file 2: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

a-d. Gene Ontology analyses showing over and under-represented gene ontologies (biological process and molecular function) in genes differentially expressed between untreated time-points. Analyses were performed using the Panther classification system [73], with a Bonferroni correction for multiple testing. e. Genes differentially expressed between IGF-1 treated and untreated samples. List includes genes identified by comparision at each timepoint, and an all v all comparison. No significant genes were identified in the day 90 analysis. (XLSX 50 kb

Additional file 6: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

FIRRE exon junctions confirmed by amplicon sequencing. All reads were mapped against hg19 without reference to transcript annotation: Amplicons using convergent primer pairs and divergent primer pairs (to amplify circRNAs only) are shown separately. Canonical junctions were mapped using MapSplice [67], circRNA (back-splice) junctions were mapped using PTESFinder [46]. For details, see methods. Exon number is according to schema in Fig. 6b. Junction position (hg19), amplicons of origin, and junction frequencies, are given for all junctions. Only splices with a frequency of 1% or higher in each amplicon, identified either by MapSplice or PTESfinder, are reported. Canonical junctions present within the current FIRRE annotation are show in blue. All others are not present within current annotation. Off target junctions (presumed to be generated by illegitimate primer binding) are also shown. Data is for confirmation of junction presence within transcripts only: Junction frequency is affected by position relative to primer, size dependent amplification bias during Nextera indexing, and size dependent bias in cluster formation/resolution efficiencies during MiSeq sequencing. (XLSX 71 kb

Additional file 7: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

Genomic co-ordinates of all annotated and novel FIRRE exons. The position of all junctions are shown with respect to hg19, together with their current annotations where appropriate. Splice acceptor and donor junctions identified by annotation-free mapping of day 0 samples against hg19 using MapSplice are shown, together with donor exons and acceptor exons, and junction numbers in brackets. Supporting evidence for each junction is colour coded. Minor / Others – additional junctions not involving annotated exons which are present at low frequency. (XLSX 12 kb

Additional file 5: of Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular

Gene Ontology analysis showing over and under-represented gene ontologies (biological process and molecular function) in parent loci of 239 significantly altered circRNAs. Analyses were performed using the Panther classification system [73], with a Bonferroni correction for multiple testing. (XLSX 11 kb

Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control

SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein-RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites

T-STAR protein regulates region-specific splicing of <i>Neurexin1-3 AS4</i> in the mouse brain.

<p>(A) <i>Neurexin</i> splicing regulation in different regions of the mouse brain (B) Schematic of the different mouse brain regions used for analysis. (C–E) Percentage splicing exclusion in different regions of the mouse brain (n = 3 mice from each genotype) measured in RNA samples from wild type (column +) and knockout (column −) mice for AS4 of (C) <i>Nrxn1</i>, (D) <i>Nrxn2</i> and (E) <i>Nrxn3</i>. The error bars correspond to the standard error of the mean. Statistical significances were calculated using a two tailed unpaired t test. No splicing exclusion was observed for <i>Nrxn3</i> in the absence of T-STAR protein in any brain region.</p

T-STAR protein concentration correlates with <i>Nrxn1-3</i> AS4 alternative splicing patterns.

<p>(A) Expression levels of T-STAR and Sam68 protein in different regions of the mouse brain were measured using Western blotting. The same filters were first probed with antisera specific for T-STAR, and then stripped and reprobed with an antisera specific for Sam68. (B–D) Levels of <i>Nrxn1-3</i> AS4 Percentage Splicing Exclusion in each brain region plotted against the ratio of T-STAR: Sam68 protein quantified from the Western blot shown in (A). The dashed line is the 95% confidence limit of the best fit line.</p

<i>Nrxn</i> exon AS4 alternative splicing control is dependent on the physiological expression of T-STAR protein even though Sam68 is co-expressed.

<p>(A) Immunolocalisation of T-STAR and Sam68 proteins in the mouse hippocampus from wild type or knockout mouse brains (Abbreviations: DG - Dentate Gyrus; and AH -Ammon's Horn). The scale bar is equivalent to 20 µm). (B) Immunolocalisation in the mouse testis. Paraffin embedded adult mouse testis sections were stained with affinity purified antibodies raised against T-STAR or Sam68 (brown staining), and counterstained with haematoxylin (blue). Abbreviations: Spg –spermatogonia; Spc –spermatocyte; Rtd –round spermatid; Spd –elongating spermatid; SC –Sertoli cell. The size bar corresponds to 20 µM. (C) Levels of <i>Nrxn1</i> and <i>Nrxn3</i> AS4 alternative splice isoforms in the testes of different mouse genotypes (n = 3 mice of each genotype) measured by RT-PCR and agarose gel electrophoresis. (D) Quantification of Percentage Splicing Exclusion in the testes of different mouse genotypes using capillary gel electrophoresis (n = 3 mice of each genotype: wild type mice <i>Khdrbs3^+/+</i> (abbreviated WT) <i>Khdrbs3^+/−</i> mice (abbreviated HET) and <i>Khdrbs3^−/−</i> mice (abbreviated KO). The p values were calculated using unpaired t tests, to determine the significance of the difference between percentage splicing exclusion levels in the wild type versus either the heterozygous <i>Khdrbs^+/−</i> mice (HET); or wild type versus the homozygous <i>Khdrbs3^−/−</i> (KO) mice. The standard error of the mean is shown as an error bar.</p

Generation of a null <i>Khdrbs3</i> allele.

<p>(A) Northern analysis of different adult mouse tissues to detect expression of <i>Khdrbs3</i> (upper panel) and small subunit rRNA (lower panel). (B) The genomic structure of the <i>Khdrbs3</i> alleles from wild type, floxed, and null mice mice were monitored using Southern blotting and the probe indicated in parts C–D. The Southern blot demonstrates that the cross with a PGK-Cre mouse successfully removed exon 2 from the genomic DNA. (C) Genomic structure of the <i>Khdrbs3^LoxP</i> conditional allele in which exon 2 of the <i>Khdrbs3</i> gene is flanked by <i>Lox</i>P sites. (D) Genomic structure of the null (<i>Khdrbs3⁻</i>) allele from which exon 2 has been deleted by Cre-mediated recombination. (E) Multiplex RT-PCR analysis of <i>Khdrbs3</i> and <i>Hprt</i> mRNA levels in different mouse tissues. The size markers are shown in nucleotides. (F) Western blot analysis of Sam68 and T-STAR protein levels in the testes of wild type and <i>Khdrbs3</i> null mice using an antibody that recognizes T-STAR and Sam68. The position of the size markers are shown in KDa.</p