UC Berkeley

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.