A role for Fkbp6 and the chaperone machinery in piRNA amplification and transposon silencing

Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other co-chaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be by-products of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification. Small RNAs were purified for preparation of high-throughput sequencing libraries. All libraries except JX56, JX57, JX36 and JX37 were generated from isolated small RNAs obtained by immunoprecipitation of the indicated proteins (Mili, Miwi2, Siwi or Ago3). JX56 and JX57 were prepared from small RNAs extracted from mouse testis. JX36 and JX37 were prepared from polyA+ RNAs extracted from the Bombyx mori cell line BmN4. Libraries prepared from mouse were prepared from mice having the indicated genotype. BmN4 cells were treated with the Hsp90 inhibitor geldanamycin (GA) before performing the immunoprecipitation in the indicated libraries. This lead to accumulation to a short species of small RNAs (16nt long) in Ago3 complexes which we called ping-pong by-product. Details can be found in Xiol et al. 2012, Molecular Cell.Piwi互作RNA（Piwi-interacting RNAs, piRNAs）对转座子的表观遗传沉默，构成了一类基于RNA的基因组防御机制。Piwi核酸内切酶可通过一种尚不明确的机制，从靶标转录本生成新的piRNA，从而扩增piRNA库。本研究鉴定出小鼠Fkbp6是该piRNA生物发生通路中的调控因子，其功能是将piRNA递送至Piwi蛋白Miwi2。缺失Fkbp6的小鼠会解除LINE1（L1）反转录转座子的转录抑制，并因Miwi2的核积累缺陷而出现DNA甲基化水平降低的表型。与其他分子伴侣辅助因子类似，Fkbp6通过其四肽重复（tetratricopeptide repeat, TPR）结构域与分子伴侣Hsp90相结合。在昆虫细胞培养模型中抑制ATP依赖的Hsp90活性，会导致Piwi复合物中出现短链反义RNA的积累。我们鉴定出这类RNA为piRNA扩增过程产生的副产物，且仅在定位于nuage颗粒的Piwi蛋白复合物中富集。我们提出，分子伴侣机器通常会主动驱逐这些抑制性RNA，从而使Piwi复合物得以循环复用，持续参与piRNA扩增过程。为制备高通量测序文库，我们对小RNA进行了纯化分离。除JX56、JX57、JX36和JX37这四组文库外，其余所有文库均由通过对指定蛋白（Mili、Miwi2、Siwi或Ago3）进行免疫沉淀所获得的小RNA构建而成。JX56与JX57文库的构建原料为从小鼠睾丸中提取的小RNA；JX36与JX37文库则取自家蚕（Bombyx mori）细胞系BmN4中提取的polyA+ RNA。所有源自小鼠的文库均使用了对应基因型的小鼠样本制备。在对应文库的免疫沉淀实验前，已预先使用Hsp90抑制剂格尔德霉素（geldanamycin, GA）处理BmN4细胞，该处理会导致Ago3复合物中积累一种长度为16nt的短链小RNA，我们将其命名为“乒乓循环副产物（ping-pong by-product）”。相关实验细节可参见Xiol等人2012年发表于《分子细胞（Molecular Cell）》的研究论文。