Fiber-type specific alternative splicing of muscle proteins is disrupted in bru1-/- IFM.

https://doi.org/10.1371/journal.pbio.3002575.s002
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posted on 2024-04-29, 17:40 authored by Elena Nikonova, Jenna DeCata, Marc Canela, Christiane Barz, Alexandra Esser, Jessica Bouterwek, Akanksha Roy, Heidemarie Gensler, Martin Heß, Tobias Straub, Ignasi Forne, Maria L. Spletter

(A) Boxplot of gene (DESeq2), exon (DEXSeq) and protein level (mass spec) expression changes between 1 d adult bru1-/- and w1118 IFM in select categories of genes including GO term “actin cytoskeleton organization,” microtubule associated genes, mitochondrial genes, GO term “muscle contraction,” RNA-binding proteins (RBPs), sarcomere proteins (SPs), and fibrillar core genes. Blue dot denotes p ≤ 0.05. (B) Venn diagram of the overlap between all significantly DE genes (purple), exons (orange), and proteins (green) between bru1-/- versus w1118 IFM in 1 d adults. (C–E) Semi-quantitative RT-PCR verification of alternative splice events in Strn-Mlck (C), wupA (D), and Mhc (E). Top: scheme of alternative isoforms with primer locations. Exon numbering in accordance with the FB2021_05 annotation. Color coding of depicted isoforms consistent with bottom panel; 3′-UTR regions in light beige. Middle: Quantification of relative expression level of splice events in tubular leg and jump (tergal depressor of the trochanter, TDT) and fibrillar IFM in control flies and in bru1M3 IFM. Error bars = SD. Bottom: representative RT-PCR gel image. (F–N’) Misexpression of GFP-tagged sarcomere proteins in bru1M3 IFM. (F, I, L) Diagrams of reporter GFP incorporation into tagged transcripts of Strn-Mlck (F), wupA (I), and Mhc-weeP26-GFP (L). Exons, magenta; 3′-UTR, tan; SA, splice acceptor; SD, splice donor; sGFP, superfold GFP. (G–N) Intensity matched single-plane confocal images from control and bru1M3 IFM at 90 h APF showing incorporation of Strn-Mlck-IsoR-GFP (G, H), wupA-GFP (J, K), and Mhc-weeP26-GFP (M, N). Strn-Mlck isoform R with sGFP tagged exon 25 is strongly expressed in wild-type IFM (G’) but absent from bru1M3 (H’). WupA with an sGFP tagged exon 3 is normally absent from wild-type IFM (J’) but gained in bru1M3 (K’). Expression of the Mhc isoform containing exon 37 and tagged in weeP26-GFP is normally restricted to early IFM development (M–M’), but is altered in bru1M3 (N–N’). GFP-tag, green; phalloidin stained actin, magenta (G, H, J, K, M, N); pseudo-coloring, GFP-intensity (compare G’ and H’; J’ and K’; M’ and N’). Scale bar = 5 μm. (O–Q) Semi-quantitative RT-PCR verification of alternative splice events in Zasp52 (O), Tm1 (P), and sls (Q). Top: schemes of alternative isoforms. Bottom: representative RT-PCR gel image. Labeled as in (C–E). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002575#pbio.3002575.s014" target="_blank">S2 Table</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3002575#pbio.3002575.s002" target="_blank">S2 Fig</a> Source Data and Gels, and the RNA-Seq data tables 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>. (TIFF)