| Proteins that harbor double stranded RNA-binding domains (dsRBDs) play functional roles in processes as diverse as RNA localization, RNA splicing, RNA editing, nuclear export of RNA and translation, yet the mechanistic basis and functional significance of dsRBDs remain unclear. To unravel this enigma, we investigated transactivation response (TAR) RNA-binding protein (TRBP), comprising three dsRBDs, which has functions in HIV replication, PKR-mediated immune response and RNA silencing. In collaborative studies using single-molecule methods, I found that TRBP exhibits an ATP-independent diffusion activity exclusively on double-stranded RNA (dsRNA) in a length-dependent manner. The first two dsRBDs of TRBP are essential for diffusion, whereas the third dsRBD is dispensable. Two homologs of TRBP, PKR activator (PACT) and R3D1-L, displayed the same behavior, implying that the ability to diffuse along dsRNA is a universal property of this protein family. Furthermore, a Dicer- TRBP complex on dsRNA exhibited dynamic diffusion, which was correlated with Dicer's catalytic activity, suggesting that the dsRNA-specific diffusion activity of TRBP contributes to enhancing small interfering RNA (siRNA) and microRNA (miRNA) processing by Dicer.;The enzyme Dicer generates 21-25 nucleotide-long dsRNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although the active site and RNA-binding regions of Dicer are functionally conserved within eukaryotes, the helicase domain has distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursors to miRNAs, whereas StDicer-2 can only cleave linear dsRNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of dsRNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, the full-length forms (but not truncated versions lacking their helicase domain) of both StDicer-1 and StDicer-2 could complement the phenotype of a mutant of a distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene, underscoring the importance of the helicase domain for Dicer function. These results further suggest that an in vivo regulatory function for the helicase domain may be conserved from fungi to humans. |
