Anti-DDX5 polyclonal antibody

The DEAD-box RNA helicase Dbp2p is highly conserved in eukaryotes and has been implicated in transcription, ribosome biogenesis, mRNP assembly, nuclear export, and long noncoding RNA (lncRNA) function. It is not understood how Dbp2p performs these seemingly unrelated biological roles. An important step toward addressing this question is the determination of cellular RNA binding sites of Dbp2p. Here, we identify transcriptome-wide RNA binding sites of Dbp2p from Saccharomyces cerevisiae using UV-crosslin king, denaturing tandem affinity purification, and next generation sequencing. We find that Dbp2p crosslinks to mRNAs and ribosomal RNAs, and markedly to noncoding RNAs, including snoRNA, snRNAs, and tRNAs. In snoRNAs, Dbp2p preferentially crosslinks at sites near the 3' ends. These sites coincide with regions where RNA-DNA hybrids (R-loops) form and with binding sites of Sen1p, another RNA helicase that functions in transcription termination and 3' processing of noncoding RNAs. We show that Dbp2p interacts in an RNA-independent manner with Sen1 p in vivo. Dbp2p crosslinks to tRNAs and other RNAs also at sites where R-loops form. Collectively, our data link Dbp2p to noncoding RNAs, Sen1p, and R-loops. The transcriptome-wide connection to R-loops provides a unifying theme for diverse cellular roles of Dbp2p.

Rationale: RNA helicases, highly conserved enzymes, are currently believed to be not only involved in RNA modulation, but also in other biological processes. We recently reported that RNA helicase DDX (DEAD-box protein)-5 is required for maintaining the homeostasis of vascular smooth muscle cells (SMCs). However, the expression and function of RNA helicase in vascular physiology and disease is unknown. Objective: To investigate the role of RNA helicase in vascular diseases. Methods and Results: We showed here that DDX-5 was the most abundant DEAD-box protein expressed in human and rodent artery, which mainly located in SMCs. It was demonstrated that DDX-5 levels were reduced in cytokine-stimulated SMCs and vascular lesions. DDX-5 knocking down or deficiency increased SMC proliferation and migration, whereas overexpression of DDX-5 prevented aberrant proliferation and migration of SMCs. Mechanistic studies revealed transcription factor GATA (GATA-binding protein)-6 as a novel downstream target of DDX-5, which directly interacted with GATA-6 and protected it from MDM (mouse double minute)-2-mediated degradation. Our ChIP assay identified a previously unreported binding of p27(Kip1) promoter to GATA-6. DDX-5 increased the recruitment of GATA-6 to p27(Kip1) promoter, which enhanced p27(Kip1) expression and maintained SMC quiescence. Finally, we showed exacerbated neointima formation in DDX-5 SMC-deficient mice after femoral artery injury, whereas overexpression of DDX-5 potently inhibited vascular remodeling in balloon-injured rat carotid artery. Conclusions: These findings provide the first evidence for a role of RNA helicase DDX-5 in the protection against SMC proliferation, migration, and neointimal hyperplasia. Our data extend the fundamental role of RNA helicase beyond RNA modulation, which provides the basic information for new therapeutic strategies for vascular diseases.