The loading of small interfering RNAs (siRNAs) and microRNAs into Argonaute proteins is enhanced
by Hsp90 and ATP in diverse eukaryotes. However, whether this loading also occurs independently of
Hsp90 and ATP remains unclear. We show that the Tetrahymena Hsp90 co-chaperone
Coi12p promotes siRNA loading into the Argonaute protein Twi1p in both ATP-dependent and
ATP-independent manners in vitro. The ATP-dependent activity requires Hsp90 and the
tetratricopeptide repeat (TPR) domain of Coi12p, whereas these factors are dispensable for the
ATP-independent activity. Both activities facilitate siRNA loading by counteracting the
Twi1p-binding protein Giw1p, which is important to specifically sort the 26- to 32-nt siRNAs to
Twi1p. Although Coi12p lacking its TPR domain does not bind to Hsp90, it can partially restore the
siRNA loading and DNA elimination defects of COI12 knockout cells, suggesting that
Hsp90- and ATP-independent loading of siRNA occurs in vivo and plays a
physiological role in Tetrahymena.
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