NMR investigation of the sliding motion of RNA helicase DDX3X along single-stranded RNA

The complex interplay between the structural formation of nucleic acids and the helicase proteins that unwind them underlies a variety of essential biological processes. Among these helicases, DDX3X is one of the most well-recognized RNA helicases, involved in diverse physiological functions such as translational regulation of gene expression. Although many structural and biochemical studies have investigated how DDX3X unwinds stable double-stranded RNA (dsRNA) into single-stranded RNA (ssRNA), the exact mechanism remains under debate. A key question is whether DDX3X processively unwinds dsRNA by translocating or sliding along ssRNA, or instead locally destabilizes dsRNA by directly binding to exposed ssRNA stretches. To gain deeper insight into the molecular mechanism of DDX3X, we used solution NMR to monitor its sliding motion on ssRNA composed of poly-C and poly-A segments. We then examined how rapidly DDX3X slides along ssRNA, a prerequisite for the processive unwinding mechanism. Our two-dimensional line-shape analyses and ZZ-exchange experiments demonstrate that DDX3X does not slide rapidly along ssRNA, with a maximum sliding rate estimated to be 10 s^-1 or slower. These findings support a model in which DDX3X unwinds dsRNA via local strand separation driven by preferential binding to ssRNA stretches, thereby providing molecular insight into its function as a non-processive helicase.