ECD functions as a novel RNA-binding protein to regulate mRNA splicing

The human ecdysoneless protein (ECD) plays an essential role in the regulation of cell cycle and cell survival. ECD has been implicated in RNA splicing through its association with the protein components of splicing complex. Here, using electrophoretic mobility shift assay and mutational analysis, we demonstrate that ECD directly binds to RNA through amino acids 135-148. Using enhanced CLIP-seq analyses, we identified a large repertoire of mRNAs bound to ECD. RNA-seq analyses revealed that ECD depletion in cells leads to widespread RNA splicing aberrations associated with alterations in gene expression. Significantly, we demonstrate that ECD mediates mRNA splicing by directly binding to RNA sequences located near splicing sites in context-dependent manner. Mechanistically, ECD associates with key proteins of the U5 snRNP complex in an RNA-independent manner and directly binds to U5 small nuclear RNA (U5 snRNA). Importantly, ECD-U5 snRNA interaction is crucial for maintaining the expression of U5-specific proteins. Furthermore, RNA binding defective mutant of ECD fails to rescue downregulated levels of U5 snRNP components or cell proliferation block induced by ECD knockout. Collectively, we provide compelling evidence that ECD regulates RNA splicing by directly associating with RNAs, and the RNA binding activity of ECD is essential for its function.