Additional file 3: of PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells

Summary of observed transcripts with their alternatively spliced 5′ and 3′ untranslated regions (UTRs). The blue rectangles on the left indicate transcripts that were detected in tissue samples whereas gray rectangles mean they were not detected. Introns are not shown for simplicity; black lines indicate that the exons were not present in the transcript. (PDF 75 kb

Additional file 4: of PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells

Transcript and protein IDs of PHACTR1 gene (221,692; ENSG00000112137). (PDF 45 kb

Additional file 5: of PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells

Zoom-in representation of PHACTR1 exon 14. The 5′-14 fraction of exon 14 is specific to the short immune-specific PHACTR1 transcript. We also illustrate the 3′ splice site used for the long and intermediate PHACTR1 transcripts. In blue is the intron between exons 13 and 14. The red arrow indicates a primer used to detect the short PHACTR1 transcript. The sequence of this primer #10 is in Additional file 1. (PDF 193 kb

Additional file 6: of PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells

Associations between genotypes at rs9349379 and PHACTR1 expression levels in human coronary arteries. (A) By qPCR using transcript-specific primers, we measured the expression of PHACTR1 transcripts in 36 human coronary arteries (hCA)(NAA = 15, NAG = 13, NGG = 8). The long PHACTR1 transcript is not expressed in hCA. (B) We used GTEx data to test the associations between rs9349379 and the expression levels of PHACTR1 exons in 122 hCA (NAA = 48, NAG = 57, NGG = 17). Here, we only show results for five exons, but association results for all PHACTR1 exons are available in Table 2. Exon 10.11 corresponds to the alternatively spliced exon located between exons 10 and 11. 5′ exon 14 corresponds to part of exon 14 that is specific to the short PHACTR1 transcript. (PDF 350 kb

Additional file 1: of PHACTR1 splicing isoforms and eQTLs in atherosclerosis-relevant human cells

List of primers used in this study. The numbers in the “Name” column refers to the primers shown in Fig. 1. All sequences are given in the 5′ to 3′ orientation. (PDF 43 kb

Frameshift indels introduced by genome editing can lead to in-frame exon skipping

<div><p>The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of “multiple of three nucleotides” exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a “multiple of three nucleotides” exon of <i>LGALS8</i> that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.</p></div

Exon skipping in <i>LGALS8</i> in human erythroblasts.

<p>(<b>A</b>) In <i>in vitro</i> differentiated human erythroblasts, three <i>LGALS8</i> mRNA isoforms are expressed. Isoform 1 includes the “multiple of three nucleotides” exon 9 (in red, 126-bp), whereas isoforms 2 and 3 do not. The nonsense variant rs2273865 (p.Leu212Ter) is located in exon 9. At this variant, the minor A-allele has a frequency of 3.5% in populations of European ancestry (ExAC). (<b>B</b>) Eight erythroblast samples are heterozygous at rs2273865 and show strong allelic imbalance (binomial <i>P</i><0.05 for all samples). Numbers in the bars indicate the numbers of reads carrying the T (green) or A (blue) allele. Differential expression of total <i>LGALS8</i> (<b>C</b>), isoform 1 (<b>D</b>), isoform 2 (<b>E</b>), and isoform 3 (<b>F</b>) between erythroblast samples homozygous TT (n = 16) and heterozygous AT (n = 8) at rs2273865. No samples homozygous for the minor allele (AA) were available. (<b>G</b>) The ratio of <i>LGALS8</i> transcripts without exon 9 over transcripts with exon 9 is higher in heterozygous AT than in homozygous TT erythroblast samples. <i>P</i>-values are calculated by linear regression correcting for cell developmental stage.</p

Frameshift indels cause in-frame exon skipping in <i>PHACTR1</i>.

<p>(<b>A</b>) <i>PHACTR1</i> expression levels measured by real-time qPCR in the parental teloHAEC cell line, an unedited clone (sg-E8N23), and clones with CRISPR-Cas9-generated frameshift indels in <i>PHACTR1</i> exon 8 (sg-E8N2 and sg-E8N16), exon 9 (sg-E9N1), and exon 10 (sg-E10N8). Data show mean and standard error of the mean from two biological replicates, done in triplicates. <i>PHACTR1</i> expression levels in sg-E8N2 is 6.2 fold greater than in the parental teloHAEC cell line (Student’s <i>t</i>-test <i>P</i> = 0.0033). (<b>B</b>) Agarose gel electrophoresis profile of the main <i>PHACTR1</i> isoforms detected in cDNA from teloHAEC cells, unedited clones, or clones with a frameshift indel. We assigned a transcript number to each of the <i>PHACTR1</i> isoform that we could Sanger sequence and align to the reference sequence. Unlabeled bands could not be assigned to <i>PHACTR1</i>. Bands in the molecular ladder correspond to 400, 500 and 700-bp. This gel is representative of three independent experiments. (<b>C</b>) Schematic diagram of all the <i>PHACTR1</i> isoforms that we identified in the different teloHAEC cell lines. Transcript numbers correspond to the bands (white numbers) in <b>B</b>. The PCR primers in exon 6 and 11 are depicted. For the isoforms expressed in edited clones, we added the corresponding nucleotide changes introduced by the frameshift indels. (<b>D</b>) Western blot of PHACTR1 in the parental, unedited and edited teloHAEC cells. The arrowhead indicates PHACTR1, lower bands are non-specific proteins recognized by the antibody. PHACTR1 is smaller in sg-E8N2, consistent with skipping of exon 8 or usage of an alternative in-frame start codon downstream of the frameshift indel. For sg-E9N1, the smaller PHACTR1 protein is consistent with skipping of exon 9. We could not detect PHACTR1 proteins in sg-E8N16 and sg-E10N8. We used GAPDH as loading control. This Western blot is representative of three independent experiments.</p

Frameshift indel can lead to exon skipping in the zebrafish <i>adgrl4</i> gene.

<p>(<b>A</b>) <i>adgrl4</i> genomic locus, TALEN target site in exon 2 of the <i>adgrl4</i> gene and a stable mutant line (Δ5) that was analyzed. (<b>B</b>) PCR analysis of <i>adgrl4</i> mRNA transcripts in 10 to 15 pooled embryo samples from control (ctrl), <i>adgrl4</i> Δ5^+/-, and <i>adgrl4</i> Δ5^-/- fishes. (<b>C</b>) Schematic representation of the different transcripts recovered from the bands (white numbers) in <b>B</b>.</p

Functional characterization of RNF186.

<p>(A) <i>RNF186</i> encodes a protein with RING domain and two transmembrane domains. E3 ubiquitin-protein ligase activity is intrinsic to the RING domain. This domain contains the disease-coding variant (A64T). (B) <i>RNF186</i> expression response to <i>S. flexneri</i> in young mice (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723.s011" target="_blank">Figure S11</a>). (C) Network building steps. Network is generated by mining multiple sources of interaction databases in Metacore that span human protein-protein, protein-DNA, Protein-RNA and protein-compounds interactions. (D) Transcriptional regulation model for <i>RNF186</i>. IL1-beta and TGF-beta 1 decrease <i>HNF4A</i> mRNA expression <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Caja1" target="_blank">[39]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Wang1" target="_blank">[41]</a>. Knockdown of retinoid X receptor, alpha (<i>RXRA</i>) down-regulates <i>HNF4A</i> gene expression; RXRA interacts with <i>HNF4A</i> gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Tomaru1" target="_blank">[24]</a>. <i>HNF4A</i> is a direct target gene of caudal type homeobox 2 (CDX2); CDX2 increases <i>HNF4A</i> mRNA expression in intestinal epithelial cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-Boyd2" target="_blank">[42]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003723#pgen.1003723-McKinneyFreeman1" target="_blank">[43]</a>. HNF4A binds promoter region of <i>HNF1A</i> and up-regulates its expression. HNF1A interacts with <i>RNF186</i> and regulates its transcription.</p