Molecular and phenotypic verification of the <i>bru1</i>^<i>M3</i> CRISPR allele.

<p><b>(A)</b> Diagram of the C-terminal region of the <i>bruno1</i> (<i>bru1</i>) locus and mRNA isoforms RA, RB, and RD (exons, purple; UTRs, yellow). Location of the RNA recognition motif domains (RRM, light red), target region of anti-Bru1 antibody (brown), target region of <i>bru1-IR</i> GD41568 hairpin (brown), location of <i>bru1</i>^<i>M2</i> construct insertion site (brown), and the sgRNAs (blue) used for CRISPR-mediated generation of the <i>bru1</i>^<i>M3</i> hypomorph allele are marked. Transgenic construct is inserted upstream of exon 18 and contains a strong splice acceptor (SA, light blue), a triple frame stop (stop, red), an SV40 polyadenylation signal (orange) and a selectable 3xP3-dsRed marker (crimson) flanked by homology arms (light tan). Exon numbering according to the annotation FB2021-05. <b>(B)</b> Whole-fly genomic PCR verifying dsRed cassette insertion in the <i>bru1</i> locus. Identity of amplified region marked on the left, band size noted on the right. Primer sequences available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002575#pbio.3002575.s016" target="_blank">S4 Table</a>. <b>(C)</b> RT-PCR to test expression of <i>bru1</i> mRNA in whole-thorax and dissected IFM. Identity of amplified region marked on the left, band size noted on the right. RpL32 used as internal control. <b>(D)</b> Diagram of the <i>bru1</i>^<i>M3</i> allele. Splicing from exon 17 is redirected into the splice acceptor (SA) of the inserted construct (red line, A), leading to early termination of the <i>bru1</i> mRNA and truncation of RRM3. Splicing from exon 17 to exon 18 is strongly reduced (dotted red line, B), and signal from 3′-UTR exon 21 is not detectable. <b>(E, F)</b> Confocal projections of 1d adult hemithoraxes showing IFM from <i>bru1</i>^<i>M3</i>/+ and <i>bru1</i>^<i>M3</i>/Df(2L)BSC407. Deficiency BSC407 covers the complete <i>bru1</i> locus. Thorax boundaries in (F), dashed line; phalloidin stained actin, gray; scale bar = 100 μm. <b>(E’–F’)</b> Single-plane confocal images of 1 d adult IFM myofibrils. Scale bar = 5 μm. <b>(G, H)</b> Quantification of sarcomere length (G) and myofibril width (H) from TEM data shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002575#pbio.3002575.g001" target="_blank">Fig 1F</a>. Boxplots are shown with Tukey whiskers, outlier data points marked as black dots. Significance determined by ANOVA and post hoc Tukey (ns, not significant; ***, p-val < 0.001). <b>(I, J)</b> Quantification of Z-disc alignment (I) and sarcomere morphology (J) defects from TEM data shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002575#pbio.3002575.g001" target="_blank">Fig 1F</a>. <i>N</i> > 20 images from 2 biological samples for each individual genotype and time point. <b>(K)</b> Dot plot showing the correlation between all detected peptide groups and their corresponding mRNA expression level in <i>bru1</i>^<i>-/-</i> versus w¹¹¹⁸ IFM (proteins with a significantly DE exon, orange; significantly DE genes, purple). The Pearson’s/Spearman’s correlation coefficients (top left corner) and regression line (blue) indicate a weak but positive correlation. Underlying data can be found in S1 and S1 Fig Source Data and Gels as listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002575#pbio.3002575.s018" target="_blank">S6 Table</a>.</p> <p>(TIFF)</p>