Woehrer Sophie L, Aronica Lucia, Suhren Jan H, Busch Clara Jana-Lui, Noto Tomoko, Mochizuki Kazufumi
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria.
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria
EMBO J. 2015 Feb 12;34(4):559-77. doi: 10.15252/embj.201490062. Epub 2015 Jan 14.
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.
在多种真核生物中，热休克蛋白90（Hsp90）和三磷酸腺苷（ATP）可增强小干扰RNA（siRNA）和微小RNA进入AGO蛋白的装载过程。然而，这种装载是否也能独立于Hsp90和ATP发生仍不清楚。我们发现，嗜热四膜虫的Hsp90共伴侣蛋白Coi12p在体外以依赖ATP和不依赖ATP的方式促进siRNA装载到AGO蛋白Twi1p中。依赖ATP的活性需要Hsp90和Coi12p的四肽重复序列（TPR）结构域，而这些因子对于不依赖ATP的活性是可有可无的。这两种活性都通过对抗Twi1p结合蛋白Giw1p来促进siRNA装载，Giw1p对于将26至32个核苷酸的siRNA特异性分选到Twi1p中很重要。虽然缺乏TPR结构域的Coi12p不与Hsp90结合，但它可以部分恢复COI12基因敲除细胞的siRNA装载和DNA消除缺陷，这表明不依赖Hsp90和ATP的siRNA装载在体内发生，并在嗜热四膜虫中发挥生理作用。
