Kai Fenzl

BACKGROUND: Pathogenic variants in RBM20 cause severe dilated cardiomyopathy. Loss-of-function variants disrupt splicing; neomorphic gain-of-function (GoF) variants also mislocalize RBM20 to cytoplasmic ribonucleoprotein granules and are associated with more aggressive disease. The mechanism by which RBM20 mislocalization drives cardiac dysfunction remains unknown. METHODS: We investigated the effects of Rbm20 GoF and loss-of-function (LoF) variants using proteomic profiling, protein solubility assays, mitochondrial respiration and calcium flux analyses, and ultrastructural imaging in mouse models. Human induced pluripotent stem cell–derived cardioids were used to validate variant-specific phenotypes. RESULTS: Rbm20 GoF, but not LoF, variants caused posttranscriptional downregulation of soluble mitochondrial proteins, including the calcium efflux regulator TMEM65 (transmembrane protein 65), and reduced solubility of mitochondrial membrane proteins. Electron microscopy revealed enlarged mitochondria with cristae disorganization. Functional assays confirmed impaired oxidative phosphorylation, reduced mitochondrial membrane potential, and abnormal calcium handling in Rbm20 GoF models. Human cardioids reproduced these findings, demonstrating that cytoplasmic mislocalization, rather than splicing deficiency, drives mitochondrial dysfunction. CONCLUSIONS: Cytoplasmic mislocalization of RBM20 disrupts mitochondrial function by reducing mitochondrial protein abundance, leading to oxidative phosphorylation failure and abnormal mitochondrial calcium handling. This mechanism distinguishes RBM20 GoF from LoF variants and may explain the more severe heart failure phenotype observed in patients with RBM20 GoF variants. These insights advance the mechanistic understanding of RBM20 -related cardiomyopathy and identify mitochondrial mRNA/protein regulation as a key node in cardiac energetics.

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20’s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20 ^+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20 ^+/+ ). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20- centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20 ^+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

Multiplex assays of variant effects have linked thousands of genotypes to fitness effects, yet we lack profound understanding of how variants impact molecular phenotypes. Here, we introduce a deep mutational scanning framework that quantifies disease-determining molecular phenotypes in human cells, allowing readouts of protein localization and splicing regulatory function at scale. Applied to the dilated cardiomyopathy (DCM)-associated protein RBM20, we profiled ~4,300 amino acid substitutions across disease-linked protein domains. Complemented by structure-function investigations of RBM20 bound to its nuclear import receptor TNPO3, we discover new variant hotspots affecting protein function. Finally, we systematically probed nuclear relocalization to identify variants that may be amenable to this therapeutic strategy. Together, we create comprehensive variant-to-function maps that predict variant impact, enhance clinical interpretation, and stratify RBM20-mediated DCM into mechanistically distinct therapeutic classes.

Protein complexes are pivotal to most cellular processes. Emerging evidence indicating dimer assembly by pairs of ribosomes suggests yet unknown folding mechanisms involving two nascent chains. Here, we show that co-translational ribosome pairing allows their nascent chains to 'chaperone each other', thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity during early translation stages is found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly-competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.

Background: Genetic diagnosis has become increasingly important to guide clinical decision making for patients with dilated cardiomyopathy (DCM). Disease-causing (P/LP) missense variants in the gene RBM20 cause a highly penetrant arrhythmogenic dilated cardiomyopathy (DCM), but the role of truncating RBM20 variants ( RBM20tvs ) is unclear. Objective: Assess the contribution of RBM20tvs to DCM. Methods: We assembled an international cohort of DCM patients with RBM20 variants and used data from the genome-first UK Biobank (UKB) to assess the etiologic fraction, natural history and penetrance of RBM20tvs . Results: The etiologic fraction of RBM20tvs in arrhythmogenic DCM was modest (0.53[0.32,0.67], p=7.5×10 ^-5 ). RBM20tv DCM patients presented to referral centers later in life than RBM20 P/LP DCM patients (53±10 vs. 34±18 years, p=4×10 ^-3 ), and were less likely to have a family history of sudden cardiac arrest (20% vs. 65%, p= 0.046) or cardiomyopathy (20% vs. 78% p=5.4×10 ^-3 ). There was no significant difference in age- and sex-adjusted incident major heart failure or arrhythmia events between RBM20tv and RBM20 P/LP DCM patients, though sex-adjusted lifetime hazard was reduced in RBM20tv DCM (HR 0.15[0.03,0.66],p=0.009). In UKB, lifetime incidence of cardiomyopathy, heart failure, or major ventricular arrhythmia diagnosis was lower in participants with RBM20tvs than in those with TTNtvs (HR 0.55 [0.36,0.84], p=5.9×10 ^-3 ). Conclusions: RBM20tvs contribute to arrhythmogenic DCM phenotypes, but confer milder disease severity alone than RBM20 P/LP variants, and reduced lifetime disease penetrance compared to TTNtvs . Their potential for additive interactions with other damaging variants should be considered in DCM patients and families.

Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20’s nuclear localization in RBM20-DCM patients.

Protein complexes are pivotal to most cellular processes. Emerging evidence indicates that pairs of ribosomes ubiquitously drive the synchronized synthesis and assembly of two protein subunits into homodimeric complexes ^1-5 . These observations suggest protein folding mechanisms of general importance enabled by contacts between nascent chains ^6,7 – which have thus far rather been considered detrimental ^8,9 . However, owing to their dynamic and heterogeneous nature, the folding of interacting nascent chains remains unexplored. Here, we show that co-translational ribosome pairing allows their nascent chains to ‘chaperone each other’, thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity in early translation stages was found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.

Co-co assembly for oligomers: Most of the human proteome forms oligomeric protein complexes, but how they assemble is poorly understood. Bertolini et al. used a ribosome-profiling approach to explore the existence of a cotranslational assembly mode based on the interaction of two nascent polypeptides, which they call the “co-co” assembly. Proteome-wide data were used to show whether, when, and how efficiently nascent complex subunits interact. The findings also show that human cells use co-co assembly to produce hundreds of different homo-oligomers. Co-co assembly involving ribosomes translating one messenger RNA may resolve the longstanding question of how cells prevent unwanted interactions between different protein isoforms to efficiently produce functional homo-oligomers. Science , this issue p. 57

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5′UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes includinga-Raf,c-RafandCdk4have long uORFs and are translated in a DENR•MCTS1 dependent manner.