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Bruno 1/CELF regulates splicing and cytoskeleton dynamics to ensure correct sarcomere assembly Drosophila flight muscles [1]

['Elena Nikonova', 'Biomedical Center', 'Department Of Physiological Chemistry', 'Ludwig-Maximilians-Universität München', 'München', 'Jenna Decata', 'School Of Science', 'Engineering', 'Division Of Biological', 'Biomedical Systems']

Date: 2024-05

Muscles undergo developmental transitions in gene expression and alternative splicing that are necessary to refine sarcomere structure and contractility. CUG-BP and ETR-3-like (CELF) family RNA-binding proteins are important regulators of RNA processing during myogenesis that are misregulated in diseases such as Myotonic Dystrophy Type I (DM1). Here, we report a conserved function for Bruno 1 (Bru1, Arrest), a CELF1/2 family homolog in Drosophila, during early muscle myogenesis. Loss of Bru1 in flight muscles results in disorganization of the actin cytoskeleton leading to aberrant myofiber compaction and defects in pre-myofibril formation. Temporally restricted rescue and RNAi knockdown demonstrate that early cytoskeletal defects interfere with subsequent steps in sarcomere growth and maturation. Early defects are distinct from a later requirement for bru1 to regulate sarcomere assembly dynamics during myofiber maturation. We identify an imbalance in growth in sarcomere length and width during later stages of development as the mechanism driving abnormal radial growth, myofibril fusion, and the formation of hollow myofibrils in bru1 mutant muscle. Molecularly, we characterize a genome-wide transition from immature to mature sarcomere gene isoform expression in flight muscle development that is blocked in bru1 mutants. We further demonstrate that temporally restricted Bru1 rescue can partially alleviate hypercontraction in late pupal and adult stages, but it cannot restore myofiber function or correct structural deficits. Our results reveal the conserved nature of CELF function in regulating cytoskeletal dynamics in muscle development and demonstrate that defective RNA processing due to misexpression of CELF proteins causes wide-reaching structural defects and progressive malfunction of affected muscles that cannot be rescued by late-stage gene replacement.

Data Availability: Raw numbers used to generate plots are available in supplementary tables and raw data files. Images of all Western blots and RT-PCR gels are provided in the raw data files. Raw data files have been deposited on the Open Science Framework (OSF, osf.io) with identifier doi: 10.17605/OSF.IO/Y7UTM and are accessible at: https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fosf.io%2Fy7utm%2F%3Fview_only%3D88ae0545828e405cbc03b3858ceb511c&data=05%7C02%7Cmaria.spletter%40umkc.edu%7C26b020ee5a1e4e9ea74808dc44102262%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C638460083306841111%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C0%7C%7C%7C&sdata=f6519Cr1hd%2BfcndNF%2BEsN6nDqKhPMyn8OtMZCEVGa6Y%3D&reserved=0 . mRNA-Seq data are publicly available from GEO with accession numbers GSE63707, GSE107247, GSE143430 and GSE205092. Whole proteome mass spectrometry data are available from ProteomeXchange with identifier PXD043308.

Here, we report that the early requirement for CELF protein function in myogenesis is conserved in Drosophila Bruno 1 (Bru1). We generated a novel CRISPR-mediated mutant in bru1 that revealed early phenotypes in cytoskeletal organization. Temporally restricted rescue and bru1 RNAi knockdown demonstrated how initial cytoskeletal defects are propagated and disrupt later steps in sarcomere growth and maturation. We further define a later requirement during myofiber maturation for bru1 to regulate sarcomere assembly dynamics, where abnormal radial growth in bru1 mutant muscle promotes myofibril fusion and the formation of hollow myofibrils. Our data moreover identify a previously uncharacterized genome-wide transition from immature to mature sarcomere gene isoform expression in IFM that is blocked in bru1 mutants. Consistent with the pleiotropic nature of the CELF misregulation phenotype, temporally restricted expression of Bru1 cannot restore myofiber function or correct structural deficits, but it does partially alleviate adult-stage hypercontraction. Our results reveal a conserved role for CELF family proteins to fine-tune sarcomere structure and function, and identify multiple distinct developmental mechanisms that contribute to the bru1 mutant phenotype in IFM.

The Drosophila IFM are an established and disease-relevant model for exploring basic mechanisms of muscle development and sarcomere assembly. Sarcomere structure is conserved, and in both insects and vertebrates, sarcomeres are built of actin thin filaments anchored at the Z-disc, myosin thick filaments anchored at the M-line, and Titin connecting filaments that span the thin and thick filaments [ 45 – 47 ]. Myosin binding to actin and filament sliding provides contractile force, while Titin influences muscle stiffness, force generation, and sarcomere length [ 48 – 51 ]. Analysis of Drosophila models of human diseases, for example, myotonic dystrophy, X-linked centronuclear myopathy, nemaline myopathy, and Duchenne muscular dystrophy, have proven informative and offer relevant insight into disease pathology [ 13 , 52 , 53 ]. Work in Drosophila also provides insight into conserved developmental mechanisms of myogenesis, including myoblast fusion, tendon attachment, sarcomerogenesis, growth, and myofibril maturation [ 54 – 56 ]. The Drosophila IFM consist of 6 dorsal-longitudinal (DLM) and 7 dorsal-ventral myofibers (DVM) in each thoracic hemisphere [ 57 , 58 ]. IFM myoblasts proliferate associated with the notum of the wing-disc, and then migrate and fuse to form IFM myotubes [ 59 , 60 ]. IFM myotubes establish tendon connections around 16 to 20 h APF [ 61 ], and then compact and undergo myofibrillogenesis around 32 h APF [ 47 , 62 , 63 ]. Sarcomeres are added to myofibrils as myofibers grow dramatically in length to span the entire thorax by 48 h APF, and from 60 h to 90 h APF sarcomeres grow to their mature size of 3.2 μm in length and 1.2 μm in width [ 47 , 64 , 65 ]. After 48 h APF, myofibrils undergo a maturation process where a switch in gene expression facilitates establishment of asynchronous and stretch-activation properties of fibrillar IFM [ 42 , 66 ]. This detailed understanding of myogenesis in a conserved genetic model system is a powerful tool that can be applied to understand how RNA regulation impacts sarcomere assembly and maturation.

The conservation of CELF protein function in myogenesis across the animal kingdom provides an opportunity to explore foundational mechanisms of RNA regulation in muscle. In zebrafish, CELF proteins are expressed in the developing mesoderm and Celf1 regulates somite development, binds to untranslated regulatory elements (UREs) and can mediate splicing of a rat α-actinin mini-gene [ 31 – 33 ]. In C. elegans, ETR-1, a CELF1 homolog, promotes muscle development through the regulation of alternative splicing and alternative 3′ exons [ 34 , 35 ]. We and others have previously shown that Bruno 1 (Bru1, Arrest), a CELF1/2 family homolog in Drosophila, acts as a splicing factor during maturation of the indirect flight muscles (IFMs) and regulates growth in sarcomere length and myosin contractility [ 14 , 36 ]. Bruno1 is also known to regulate posterior localization and translation repression of oskar and gurken mRNAs in Drosophila embryos, helping to establish the anterior-posterior axis during embryogenesis [ 37 – 41 ]. In IFM, Bru1 expression is activated by the master regulator of the fibrillar muscle fate Spalt major (Salm), and hundreds of IFM-specific splice events in structural genes are lost after bru1 RNAi knockdown [ 14 , 36 ]. Although the direct targets of Bru1 and detailed molecular mechanisms that contribute to the Bru1 phenotype are not known, loss of a Bru1-regulated, IFM-specific isoform of Stretchin-Myosin light chain kinase (Strn-Mlck) is sufficient to induce hypercontraction, short sarcomeres, and loss of myofibers [ 14 , 42 ]. Bru1 genetically interacts with RNA-binding protein Rbfox1 in IFM, resulting in complete loss of sarcomeric structure when both proteins are knocked-down and mirroring a regulatory interaction observed in mammals [ 27 , 43 ]. Although Bru1 levels peak early in IFM development and are down-regulated in adult flies [ 43 ], all reported phenotypes for Bru1 affect later steps in sarcomere maturation after 48 h after puparium formation (APF) [ 14 , 36 , 43 , 44 ]. The question therefore arises if Bru1 has a function during early stages of IFM formation, congruent with the role of CELF1/2 in fetal muscle in vertebrates, or if CELF function in Drosophila muscle is mechanistically distinct.

CUG-BP- and ETR-3-like factor (CELF) family RNA-binding proteins (also known as Bruno-like proteins) are important regulators of RNA processing. CELF proteins contain 3 highly conserved RNA recognition motif (RRMs) domains that are jointly involved in binding to GU-rich recognition elements in RNA [ 16 , 17 ]. They regulate diverse steps in RNA processing, from alternative splicing to mRNA trafficking, stability, decay, and translation [ 18 – 20 ]. In striated muscles, CELF proteins are involved in regulating developmental transitions in alternative splicing. CELF1 and CELF2 promote embryonic splicing patterns in vertebrate heart and skeletal muscle [ 21 , 22 ], for example, promoting inclusion of cardiac troponin T (cTNT) exon 5 in embryonic heart affecting calcium sensitivity and contractility in mouse and chicken [ 23 ]. CELF1/2 levels are down-regulated 10-fold as heart and skeletal muscle mature [ 21 , 22 ], and overexpression of CELF1 during mouse heart development affects nearly 30% of developmental-associated splicing changes, largely promoting reversion to the embryonic splicing pattern [ 24 ]. While CELF1/2 are down-regulated in muscle development, Muscleblind-like family proteins MBNL1 and MBNL2 are in contrast up-regulated and promote mature splicing and polyadenylation patterns [ 21 , 25 , 26 ]. CELF1/2 and MBNL1/2 antagonistically co-regulate the alternative splicing of hundreds of exons in developing muscle [ 24 , 27 ]. The physiological relevance of this regulatory interaction is illustrated by the severity of muscle phenotypes in myotonic dystrophy type I (DM1) patients, where sequestration of MBNL1 through binding to a repeat expansion in the DMPK gene results in PKC-mediated stabilization and increased expression of the CELF1 protein [ 28 , 29 ], and a corresponding reversion from mature to embryonic isoform expression patterns [ 21 , 25 , 28 , 30 ]. Thus, CELF proteins are a key component of the RNA regulatory network that defines muscle structure and contractile ability during myogenesis.

Alternative splicing plays a key role in shaping the diverse contractile and morphological characteristics of different striated muscle fiber types [ 1 , 2 ]. For example, the heart expresses short splice isoforms of Titin that contribute to the high passive resting stiffness of cardiomyocytes [ 3 , 4 ], while skeletal muscles with a lower passive resting stiffness express longer and more flexible Titin isoforms [ 5 , 6 ]. Fast and slow muscle fibers express different isoforms of Troponin I (TnI) and Troponin T (TnT), resulting in differences in Ca 2+ sensitivity and contractile dynamics [ 7 ]. The fiber-type-specific expression patterns of hundreds of exons are established during development, with transitions to mature isoforms promoting acquisition of fiber-type characteristic contractile properties [ 8 – 10 ]. Although the functional differences between most splice isoforms are still unknown, misregulation of alternative splicing and isoform expression in muscle diseases such as dilated cardiomyopathies and myotonic dystrophies contributes to contractile dysfunction [ 11 – 13 ], highlighting the importance of RNA regulation to normal muscle function. Even different muscle fiber types in model organisms such as Drosophila melanogaster have distinct alternative splicing profiles [ 14 , 15 ], indicating that the regulation of alternative splicing and structural isoform expression plays a conserved role in fine-tuning muscle structure and contractile properties.

Results

To investigate the function of Bru1 in Drosophila IFM development, we generated a new CRISPR allele that we refer to as bru1M3. bru1M3 is a truncation allele resulting from the integration of a splice-trap cassette upstream of bru1 exon 18 (S1A and S1B Fig) that results in a near complete loss of detectable bru1 mRNA and protein expression (S7A, S7C and S7C’ Fig). The splicing of bru1 transcripts is redirected into the splice acceptor of the cassette instead of into exon 18, generating an early termination that effectively deletes the most C-terminal 88 amino acids in RRM3 of all bru1 isoforms (S1C and S1D Fig). Based on phenotypes reported for the aretQB72 allele (EMS-induced stop at position 404) [14,38,67], as well as point mutations in RRM3 at positions 521 and 523 [39], bru1M3 is predicted to be a phenotypic null allele. Like other bru1 alleles [38,67], bru1M3 is male and female sterile. Consistent with the reported adult IFM phenotype of bru1 RNAi knockdown (bru1-IR) [14,36] and bru1 alleles bru1M2 [43], bru1M1 [13], bru1QB72, bru1PD41, and bru1PA62 [14], we found that bru1M3 mutants are flightless and display a loss of myofibril and sarcomere architecture (Fig 1A–1C). We further confirmed the specificity of this phenotype over deficiency Df(2L)BSC407, which covers the bru1 locus (Fig 1B and 1C, S1E, S1E’, S1F and S1F’ Fig). Together, these findings validate the nature and specificity of the bru1M3 allele and provide independent confirmation of a function for Bru1 in IFM development.

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TIFF original image Download: Fig 1. bru1 mutant flight muscle displays misregulated sarcomere protein expression and progressively severe phenotypes during myofibril maturation. (A) Single-plane confocal images from thorax hemi-sections of w1118 and bru1M3 at 48 hour (h), 60 h, 72 h, 80 h, 90 h APF and 1 d adult flies. Phalloidin stained actin, gray; scale bar = 5 μm. (B) Quantification of flight ability. N > 30 flies for each genotype. (C) Quantification of myofiber phenotypes. N > 40 myofibers from 10 flies for each genotype. (D, E) Quantification of the sarcomere length (D) and myofibril width (E) from (A). Sarcomeres in control w1118 flies grow significantly in length from 2.0 ± 0.1 at 48 h APF to 2.9 ± 0.2 μm at 90 h APF (ANOVA, p < 0.001), while sarcomeres in bru1M3 do not (2.1 ± 0.1 to 2.2 ± 0.3 μm; ANOVA, p = 0.96). At 60 h APF, bru1M3 myofibrils are significantly wider than in wild type (0.99 ± 0.2 versus 0.66 ± 0.04 μm, ANOVA, p < 0.001). bru1M3 myofibrils significantly increase in width from 0.48 ± 0.06 at 48 h APF to 1.03 ± 0.26 μm at 90 h APF (ANOVA, p < 0.001). From 80 h APF to 1 d adult, bru1M3 sarcomeres shorten (2.3 ± 0.2 to 2.1 ± 0.2 μm; ANOVA, p = 0.009), while w1118 sarcomeres grow (2.5 ± 0.2 to 3.3 ± 0.1 μm; ANOVA, p < 0.001). Myofibril width increases more from 80 h APF to 1 d adult in bru1M3 (1.1 ± 0.3 μm to 2.4 ± 0.4 μm; ANOVA, p < 0.001) than in w1118 (0.76 ± 0.04 to 1.0 ± 0.1 μm; ANOVA, p = 0.002). 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 < 0.01; ***, p < 0.001). (F) TEM images of w1118 and bru1M3 sarcomere ultrastructure at 48 h, 60 h, 72 h, and 90 h APF. Defects in bru1M3 are already apparent at 48 h APF. Z-discs, “Z”; myofibril splitting and discontinuous Z-discs, white arrows; cytoplasm or mitochondrial inclusions, white asterisks; scale bar = 1 μm. (G) Quantification of Z-disc integrity in (F). N > 20 single planes for each individual genotype and time point. (H, I) mRNA-Seq volcano plots of DESeq2 gene expression (H) and DEXSeq exon use (I) changes in 1 d adult bru1M3-/- versus w1118 IFM. SPs are notably affected (red dots). Gray boxes denote a threshold of abs(log 2 fold-change) ≥ 1 and p ≤ 0.05, with significant events colored blue. (J) Volcano plot of peptide group expression (J) changes in bru1-/- IFM from 1 d adults. Gray boxes denote a threshold of abs(Difference) ≥ 1 and p ≤ 0.05, significant peptides are colored blue. (K) Heatmap of select significantly enriched biological process GO terms in the DE genes, exons and proteins. (L) Dot plot of the correlation between significantly DE peptide groups and their corresponding mRNA expression level in bru1-/- versus w1118 IFM. Proteins with a significantly DE exon (DEXSeq) are colored orange, and those significantly DE at the gene level (DESeq2) are colored 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 Table and Fig 1 Source Data files as listed in S6 Table. APF, after puparium formation; DE, differentially expressed; GO, gene ontology; IFM, indirect flight muscle; SP, sarcomere protein; TEM, transmission electron microscopy. https://doi.org/10.1371/journal.pbio.3002575.g001

Misregulation of gene expression and splicing lead to protein expression defects in bru1M3 muscle We next investigated the molecular phenotype underlying the myofibril defects observed in bru1M3 IFM. We performed mRNA-Seq and whole proteome mass spectrometry on IFM dissected from 0- to 24-hour-old (1 d adult) wild-type w1118 or bru1 mutant flies, to evaluate changes on both the RNA and protein levels (Fig 1H–1J and S1 Table). A differential expression analysis with DESeq2 revealed hundreds of significant changes in gene expression in the mRNA-Seq data (Fig 1H). Up-regulated genes were enriched for biological process gene ontology (GO) terms such as “muscle attachment,” “sarcomere organization,” “actin cytoskeletal organization,” “actin filament capping,” and “establishment of RNA localization” (Fig 1K). Down-regulated genes were in contrast enriched for terms such as “translation,” “cation transport,” and “oxidation-reduction process.” Using DEXSeq, we further detected hundreds of significant changes in exon use in bru1M3 versus w1118 IFM (Fig 1I), reflecting changes in alternative splicing as well as alternative promoter use. Notably, both up-regulated and down-regulated exons were enriched for GO terms such as “sarcomere organization,” “actin cytoskeleton organization,” “muscle contraction,” and “calcium-mediated signaling” (Fig 1K). This likely reflects isoform switches in structural genes, as for example sarcomere proteins (SPs) display both up- and down-regulated exons (Fig 1I), which we investigate in more detail below. Interestingly, on the gene level, SPs are mostly up-regulated in bru1M3 versus w1118 IFM, potentially reflecting transcriptional compensation in response to changes in isoform use (Fig 1H). We conclude that loss of Bru1 function leads to changes in both gene expression and alternative splicing. We complimented our transcriptomic data with proteomics from 1 d adult IFM to evaluate if mRNA-level changes translate to altered protein expression. We grouped detected peptides into protein groups, such that peptides from the same gene that are unique to different protein isoforms form distinct protein groups. Analysis of the proteomics data revealed significant changes in the expression of hundreds of protein groups, with a bias toward down-regulation (Fig 1J). Down-regulated proteins were enriched in GO terms such as “muscle system process,” “muscle contraction,” “electron transport chain,” and “oxidation-reduction process,” while up-regulated proteins were enriched in “tissue development” and “intracellular receptor signaling process” (Fig 1K). As in the exon use analysis, we see up- and down-regulation of different sets of protein groups from SPs (Fig 1J). We then tested if there is a relationship between the changes in gene expression at the mRNA and protein level, and observed a weak but positive correlation for all significantly changed protein groups (Pearson’s R2 = 0.34, Spearman’s R2 = 0.41) (Fig 1L). Interestingly, we saw that both changes in gene expression as well as changes in exon use correlated with differential expression on the protein level (Fig 1L and S1K Fig). We also noted that different categories of genes show distinct patterns of regulation on the mRNA and protein level. Cytoskeletal genes such as SPs, genes involved in actin cytoskeleton organization, and microtubule-associated genes tend to be up-regulated at the gene level, but show both up- and down-regulation in the DEXSeq and proteomics data (S2A Fig). Mitochondrial and fibrillar core genes [14], by contrast, have up- and down-regulated exons, but are down-regulated on both the gene and protein level (S2A Fig). We conclude that Bru1-mediated changes to the IFM transcriptome are complex and indeed alter both protein and protein isoform expression.

A developmental switch to mature splice isoforms is blocked in IFM lacking Bru1 Based on the developmental transition in gene expression, we hypothesized that a similar transition in alternative splicing exists in wild-type IFM. We next investigated if such a splicing transition exists, and if it is disrupted in IFM lacking Bru1. The Biological Process GO terms enriched in the hundreds of DE exons at all 4 time points in our mRNA-Seq time course (Fig 4A) included “actin cytoskeleton organization,” “actin filament-based process,” “actomyosin structure organization,” “sarcomere organization,” and “cytoplasmic translation” (Fig 4C). We therefore started by looking at exon use dynamics in core fibrillar muscle genes and SPs, as several mature, IFM-specific protein isoforms have been reported in these categories [14,66,79,80]. In total, at 24 h, 30 h, or 72 h APF or at 1 d adult, we saw significant changes in 222 exons from 73 core fibrillar muscle genes and 413 exons from 56 SPs in bru1-IR IFM (S4D Fig). Strikingly, exons that were up-regulated in bru1-IR IFM were down-regulated in control IFM between 24 h APF to 1 d adult, while exons that were down-regulated in bru1-IR IFM were up-regulated in control IFM between 24 h APF to 1 d adult. To expand beyond SPs and fibrillar genes, we next identified a set of 91 exons that are significantly misregulated in at least 3 of 4 time points in bru1-IR IFM. These exons became more strongly misregulated as development proceeds and belonged to genes encoding ribosomal subunits, microtubule-associated genes, SPs, contractile fiber, and myosin complex genes (Fig 4D). When we looked at the temporal change in use of these exons in control muscle from 24 h APF to 1 d adult, we found that all of the exons up-regulated in bru1-IR IFM are normally down-regulated in 1 d adult muscle. Likewise, the exons down-regulated in bru1-IR IFM are normally up-regulated between 24 h APF and 1 d adult in control IFM (Fig 4D). We then identified all exons in our mRNA-Seq time course that are significantly regulated between 24 h APF and 1 d adult in control IFM (Fig 4F). We observed a clear temporal switch in exon use, reflecting mainly alternative splice events and alternative 3’ UTRs, but also alternative promoter use (S3 Table). Strikingly, when we plotted the temporal change in use of these exons in bru1-IR IFM, we observed a reduction in coordinated developmental regulation. We confirmed these mRNA-level changes at the protein level for wupA, Kettin, and Clip190 using GFP-tagged reporters under the control of endogenous regulatory elements (S5A and S5C–S5E Fig). This analysis reveals that loss of Bru1 results in a block in the temporal shift in exon use during IFM development, including increased expression of tubular-preferential isoforms. Thus, the molecular defect that underlies the myofibril growth and hypercontraction defects observed during myofibril maturation in bru1-IR and bru1M3 IFM is a failure to transition to expression of mature, muscle-type-specific splice isoforms.

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