A new study led by Wei Guo, associate professor in animal and dairy sciences, has uncovered a new mechanism that may help explain how certain mutations in the cardiac splicing factor RBM20 contribute to severe dilated cardiomyopathy. Guo’s lab focuses on the role of RBM20 in striated muscle biology, including cardiomyopathies, heart failure, and muscle regeneration. Their new study suggests that disease-causing RBM20 mutations may affect the heart via two separate processes.
RBM20 is an RNA-binding protein that plays an important role in regulating alternative RNA splicing in the heart. Under normal conditions, RBM20 is primarily located in the nucleus of heart muscle cells, where it forms nuclear speckles. However, certain disease-causing mutations disrupt RBM20’s ability to enter the nucleus, causing the protein to accumulate in the cytoplasm and form abnormal molecular condensates. Patients carrying these mutations can develop a particularly severe form of dilated cardiomyopathy, characterized by enlargement and weakening of the heart and an increased risk of arrhythmias and heart failure.
Previous studies from the Guo lab and others have shown that these mutations cause a loss of RBM20’s normal function in the nucleus, leading to abnormal splicing of its target genes. Increasing evidence, however, suggests that the abnormal cytoplasmic RBM20 granules themselves may also contribute to disease through a separate gain-of-function mechanism. This raised several important questions: What proteins are contained within normal RBM20 nuclear speckles and disease-associated cytoplasmic granules? Do these abnormal granules interfere with the function of other RNA-binding proteins? And could they disrupt RNA splicing beyond the genes normally regulated by RBM20?
In the new study, published in Nucleic Acids Research, Yanghai Zhang, a postdoctoral researcher in the Guo lab, and colleagues used proximity-labeling proteomics to identify proteins associated with normal RBM20 nuclear speckles and disease-associated cytoplasmic RBM20 granules. They found that the two compartments contain different groups of proteins, although several proteins involved in RNA processing were associated with RBM20 in both locations.
Among these potential interacting proteins, the team focused on two important cardiac splicing factors, CELF1 and MBNL2. They found that RNA-splicing events normally regulated by CELF1 and MBNL2 were altered in mouse models carrying disease-causing RBM20 mutations that form cytoplasmic granules. Importantly, similar changes were not observed in models that lost normal RBM20 nuclear function but did not form cytoplasmic granules. This suggests that these splicing abnormalities are not simply caused by the loss of RBM20 function in the nucleus but may instead be linked to the formation of abnormal RBM20 granules in the cytoplasm.
To further test this connection, the team used a previously established antisense oligonucleotide approach to reduce RBM20 expression and decrease cytoplasmic granule formation. After RBM20 granules were reduced, abnormal splicing of MBNL2-regulated genes showed a trend toward partial recovery, while CELF1-regulated genes did not show clear improvement. These findings further support the idea that cytoplasmic RBM20 granules can interfere with other splicing factors and disrupt the RNA-splicing programs they normally control.
Together, the findings suggest that disease-causing RBM20 mutations may affect the heart in two ways. First, they reduce RBM20’s normal splicing activity in the nucleus. Second, the abnormal RBM20 granules that form in the cytoplasm may interfere with other splicing factors, including CELF1 and MBNL2, and disrupt a broader network of RNA splicing.
This study expands our understanding of how RBM20 mutations contribute to severe cardiomyopathy and suggests that abnormal cytoplasmic RBM20 granules may represent a potential target for future therapies.