A novel RNA-binding activity of ECD contributes to U5 snRNP stability and pre-mRNA splicing

Human ecdysoneless protein (ECD) plays an essential role in regulating cell cycle progression and cell survival. ECD has previously been implicated in RNA splicing through its association with spliceosomal proteins. Here, using electrophoretic mobility shift assay, fluorescence polarization assays, and mutational analysis, we demonstrate that ECD directly binds to RNA. Enhanced CLIP-seq analysis identified a broad repertoire of mRNAs bound to ECD in cells. RNA-seq analyses revealed that ECD depletion leads to widespread splicing aberrations and altered gene expression. ECD binding to RNAs was enriched near splice sites, and a substantial fraction of ECD-bound transcripts exhibited splicing defects upon ECD depletion. ECD associates with and stabilizes the U5 small nuclear ribonucleoprotein (snRNP) complex specific proteins. While depletion of ECD reduced the levels of key U5-specific proteins, these proteins exhibited an increased association with the R2TP complex in knockout cells. Notably, we found ECD to directly bind to U5 snRNA, and an RNA binding defective mutant of ECD (Δ135-148) failed to rescue the reduced levels of U5-specific proteins or the proliferation defect induced by ECD depletion. Collectively, these findings demonstrate that ECD binds to RNAs, including the U5 snRNA, and that RNA-binding is required for ECD to stabilize the U5 snRNP and for cellular functions.