ECD, a novel U5 snRNA binding protein regulates RNA splicing [RNA-Seq, RIP-Seq]

The human ecdysoneless protein (ECD) plays a pivotal role in cellular and physiological processes, including the regulation of the cell cycle, cell survival, and embryogenesis. More recently, ECD has been implicated in RNA biogenesis through its regulatory effects on mRNA nuclear export and RNA splicing. In the current study, we identified the mechanism of ECD’s function in RNA splicing. We demonstrate a novel role of ECD as an RNA-binding protein and, through mutational analyses, we show that it utilizes its N-terminal region to interact with RNAs. By employing unbiased approaches such as eCLIP-seq and RNA-seq analyses in hTERT-immortalized mammary epithelial cells (76NTERT), we demonstrate that ECD directly binds to RNA and predominantly utilizes its RNA-binding abilities to regulate RNA splicing. This regulation occurs either through direct binding of ECD to RNA sequences located near splicing sites, one of the top ECD binding motifs identified being purine-rich exonic splicing enhancers or by influencing the biogenesis of the U5 small nuclear ribonucleoprotein particle (U5 snRNP) complex via its direct interaction with U5 snRNA. Notably, this interaction not only maintains the expression of U5 snRNA but also is essential for maintenance of protein components of the U5 snRNP complex, such as PRPF8 and EFTUD2. Consistently, ECD KO in 76NTERT cells lead to widespread aberrant splicing of numerous RNAs leading to alteration in expression of several genes. Importantly, ECD KO or the loss of its RNA-binding function resulted in the downregulation of U5 snRNP components, which functionally culminates into cellular proliferation block. Significantly, full-length ECD but not its RNA binding site mutant failed to rescue cell proliferation blocks upon ECD KO. Collectively, we provide compelling evidence that ECD regulates RNA splicing by directly associating with U5 snRNP and regulating U5snRNP complex to maintain cell proliferation and survival.