A Tetrahymena Piwi Bound to Mature tRNA 3' Fragments Activates the Exonuclease Xrn2 for RNA Processing in the Nucleus. Tetrahymena thermophila

Emerging evidence suggests that Ago/Piwi proteins function in the nucleus as well as the cytoplasm to control gene expression, but the mechanisms they employ in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. We show that Twi12 interacts with the exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2 in vivo, as well as activate its exonuclease activity in vitro. When Twi12 or Xrn2 are depleted, pre-rRNA processing intermediates accumulate and mature rRNA levels decline. RNA polymerase I and II (RNAP I and II) transcripts also accumulate. Twi12 function depends on small RNA (sRNA) binding, which is required for its nuclear import. Twi12-bound sRNAs are Dicer-independent 3' tRNA fragments that we propose may not be involved in sequence-specific base pairing to targets. Our findings suggest a role for tRNA fragments and an Ago/Piwi protein in global control of gene expression through interaction with a conserved exonuclease. Overall design: One library is analyzed here: small RNAs associated with ZZF-tagged Twi12. Twi12 is the full-length version, which is extended by 17 amino acids at the N-terminus compared to the previously-reported Twi12. Specifically, the 16-22 nt reads were analyzed here.

越来越多的证据表明，Ago/Piwi蛋白（Ago/Piwi proteins）不仅可在细胞质中发挥功能，也可定位于细胞核以调控基因表达，但其在细胞核内的作用机制仍未得到充分阐明。嗜热四膜虫（Tetrahymena thermophila）的Ago/Piwi蛋白Twi12是细胞生长所必需的，且定位于细胞核内发挥功能。本研究发现，Twi12可与核酸外切酶Xrn2（exonuclease Xrn2）相互作用。Twi12可在体内稳定Xrn2并介导其亚细胞定位，同时在体外激活其核酸外切酶活性。当Twi12或Xrn2被耗竭时，核糖体RNA前体（pre-rRNA）的加工中间产物会发生积累，成熟核糖体RNA（rRNA）的表达水平则会下降；RNA聚合酶I与II（RNAP I和II）的转录本同样会出现积累。Twi12的功能依赖于小分子RNA（small RNA, sRNA）的结合，而这一结合是其完成核输入的必要条件。与Twi12结合的sRNAs为不依赖Dicer的3'端转运RNA片段（tRNA fragments），我们推测其可能并不通过序列特异性碱基配对与靶标结合。本研究结果表明，转运RNA片段与Ago/Piwi蛋白可通过与保守型核酸外切酶相互作用，参与基因表达的全局调控。实验整体设计：本研究仅分析了一个文库，即与ZZF标签融合的Twi12相结合的小分子RNA文库。本研究所用的Twi12为全长版本，与既往报道的Twi12相比，其N端额外延伸了17个氨基酸残基。具体而言，本研究分析了长度为16~22 nt的测序读段（reads）。