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. 2015 Feb;16(2):178-91.
doi: 10.15252/embr.201439791. Epub 2014 Dec 22.

The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle

Maria L Spletter  1 , Christiane Barz  1 , Assa Yeroslaviz  1 , Cornelia Schönbauer  1 , Irene R S Ferreira  1 , Mihail Sarov  2 , Daniel Gerlach  3 , Alexander Stark  3 , Bianca H Habermann  1 , Frank Schnorrer  4
Affiliations

The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle

Maria L Spletter et al. EMBO Rep. 2015 Feb.

Abstract

In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.

Keywords: Arrest; Drosophila; alternative splicing; flight muscle; myofibril.

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Figures

Figure 1
Figure 1
Expression of Muscle-type-specific RNA and protein isoform depends on salm

  1. Wild-type fibrillar IFMs (A) and tubular leg (B) and jump muscles (TDT) (C). Knock down of salm leads to tubular conversion of the IFMs (D), leg and jump muscles are unaffected (E, F).

  2. mRNA-Seq read counts showing expression of Strn-Mlck (G–M) and kettin isoforms (N–T) from wild-type IFMs, leg muscle and jump muscle as well as salm-IR or salmFRT conditional mutant IFMs (G, N), and by genomic GFP-tagged isoform markers in Strn-Mlck (H–M) and kettin (O–T). Note that the IFM-specific expression of Strn-Mlck-IsoR depends on salm (compare H and K), whereas the tubular muscle-specific Kettin-IsoA/D is gained in IFMs upon loss of salm (compare O and R). Insertion of the GFP tag is indicated by green arrows in G and N. Hairpin sequence of Strn-Mlck-IR and the MiMIC insertion site are marked in G. Note the fibrillar-specific exons marked with green boxes (G, N) and the tubular-specific exons in Strn-Mlck marked by a red box (G).

Data information: Black arrows indicate direction of transcription. Scale bars are 5 μm, and all images were cropped to the same size.
Figure 2
Figure 2
Systematic identification of salm-dependent fibrillar and tubular muscle-specific exons

  1. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DESeq2) genes whose log2-fold changes are greater than 2 (log2FC > 2). Note that expression of 133 IFM-specific genes is salm dependent.

  2. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DEXSeq) exons with log2FC > 2. Note that expression of most IFM-specific exons is salm dependent (n = 794). These exons combined with the genes in (A) define the core group of fibrillar-specific genes.

  3. REVIGO treemap of GO component analysis of the 703 core fibrillar genes versus all expressed genes showing enrichment for muscle, mitochondrial, and cytoskeletal terms.

  4. Tree of selected GOrilla GO component terms highlighting enrichment of muscle structural components.

  5. Hierarchical clustering of log2FC of all 319 exons from 53 sarcomeric genes that are significantly differentially expressed (P-value < 0.05, DEXSeq) comparing their expression in IFMs to entire legs, jump muscles, salm-IR IFMs or aret-IR IFMs. Exons cluster into ‘fibrillar exons’ (shown in reds) which are IFM specific and mostly salm dependent and ‘tubular exons’ (shown in blues). Black dots on right demark location of individual sarcomeric gene exons in the heatmap. Note that individual sarcomeric genes have both tubular- and fibrillar-specific exon expression.

Figure 3
Figure 3
IFM-specific expression and nuclear localization of Aret

  1. Developmental mRNA-SEQ analysis of aret expression in IFMs, leg muscle and jump muscle. aret mRNA is strongly expressed at 30 h APF and expression continues in adult IFMs, but not in leg or jump muscles. aretIR hairpin sequences are indicated.

  2. Aret protein localizes to IFM nuclei (B) but not to leg muscle (C) or jump muscle nuclei (D) in adults. Both aret mRNA and protein expression depend on salm (A, E). Scale bar are 5 μm.

Figure 4
Figure 4
aret is required for flight muscle function and fiber integrity

  1. Flight tests of various aret-specific RNAi hairpins and trans-heterozygous aret mutant combinations.

  2. Hemithoraces (B–D) and IFM myofibrils (E–G) from young (day 1) wild-type (B, E), aret-IR (C, F), or aret mutants (D, G). Note the thinner or ruptured IFM fibers upon aret removal (C, D, red arrow heads) and the shorter or entirely disrupted sarcomeres (F, G compare to E). Scale bars are 100 μm in (B–D).

  3. Hemithoraces (H–J) and IFM myofibrils (K–M) of aged adults (5–7 days) from wild-type, aret-IR, or aret mutants. Note the severe IFM fiber disruption (I, J, red arrow heads) and the severe myofibril and sarcomere defects upon aret removal (L, M compared to K). Leg muscles are unaffected (N–P). Scale bars are 100 μm (H–J), and 5 μm (E–G) and (K–P).

Figure 5
Figure 5
Aret shuttles between the cytoplasm and nucleus during IFM development

  1. Aret protein is concentrated in sub-nuclear locations of the larval IFM template muscles at 14 h APF (red arrow heads in A). Aret protein is barely detectable at 17 h APF and its levels increase until 60 h APF, during which time Aret is located in close proximity to the nuclei and throughout the IFM cytoplasm (B–F). Aret protein is found in the nuclei at 72 h APF (G) and in adult IFMs (H). Scale bars are 5 μm.

  2. Co-stain of Aret and nuclear Lamin reveals some Aret in the nuclei at 48 h APF (I), whereas most of Aret is nuclear at 72 h APF (J). Scale bars are 5 μm.

Figure 6
Figure 6
aret is essential for myofibril maturation and sarcomere elongation

  1. Developing myofibrils and sarcomeres in wild-type (A–D) and aret-IR pupae (E–H) stained with phalloidin. Intensity plots 10 μm in length within one representative myofibril (A'–H'). Regular sarcomeres of about 2 μm at 48 h APF in wild-type (B) mature to about 3.3 μm at 90 h APF (D). aret-IR myofibrils and sarcomeres are present at approximately the normal length at 48 h APF (F) but fail to elongate until 90 h APF (G, H). Scale bars are 5 μm.

  2. Quantification of sarcomere length in wild-type (blue) and aret-IR (red), ***P < 0.001, unpaired Student's t-test. Standard deviation is shown.

Figure 7
Figure 7
aret regulates fibrillar muscle-specific alternative splicing

  1. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DESeq2) genes with a log2FC > 2 between aret-IR and salm-IR. 51 genes are co-regulated by Salm and Aret.

  2. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DEXSeq) exons with log2FC > 2 between aret-IR and salm-IR. Note that expression of 78.6% (1119/1423) of Salm-dependent exons is also Aret dependent.

  3. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DESeq2) genes with a log2FC > 2 between IFM:aret-IR, IFM:salm-IR, IFM:leg, and IFM:jump muscle. Only 24 fibrillar-specific genes are co-regulated by Salm and Aret.

  4. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DEXSeq) exons with log2FC > 2 between IFM:aret-IR, IFM:salm-IR, IFM:leg, and IFM:jump muscle. 747 fibrillar-specific exons co-depend on Aret and Salm.

  5. Correlation plot of the log2FC IFM:salm-IR versus IFM:aret-IR. All significantly differentially expressed exons (n = 5939, P-value < 0.05, DEXSeq) are plotted in black, while sarcomeric protein exons are plotted in red. Pearson's correlation coefficients for all exons (black) and the sarcomeric exons subset (red) are indicated. Note that many exons are co-regulated by both Salm and Aret and that Aret promotes both inclusion and exclusion of exons.

  6. Venn diagram comparing significantly differentially expressed (P-value < 0.05, DEXSeq) exons with log2FC > 2 between WT IFM:aret-IR IFM at 30 h APF, 72 h APF and in 1-d adults. Notably, 781 exons (491 uniquely) are regulated at the adult time point, while only ˜300 exons are regulated at each developmental time point.

Figure 8
Figure 8
Age-dependent aret-IR fiber degeneration is caused by hyper-contraction

  1. Hemithoraces of 90 h APF pupae (A, D, G, J), 1-day adults (B, E, H, K) and 5–7 days aged adults (C, F, I, L). Wild-type IFM fibers remain intact in aged adults (A–C), whereas aret-IR fibers are successively ruptured and lost (D–F, red arrow heads). This fiber loss is entirely suppressed upon removal of Mhc function from aret-IR IFMs using the Mhc [10] allele (G–L). Scale bars are 100 μm.

  2. Hemithoraces of IFM-specific Strn-Mlck knock down 90 h APF pupae (M), 1 day (N) and 5–7 days adults (O) and Strn-Mlck MiMIC [MI02893] 90 h APF (P), 1 day (Q) and 5–7 days adults (R). Note the progressive fiber degeneration upon aging (M–R, red arrow heads). Scale bars are 100 μm.

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