(Background) Piwi-interacting RNAs (piRNAs) are predominantly expressed in germline cells and function in transposon silencing. Unlike other small RNAs, piRNAs are processed from long single-stranded precursor piRNAs (pre-piRNAs) independently of Dicer. At the final step in piRNA biogenesis, 3’ ends of pre-piRNAs are trimmed by putative exonuclease “Trimmer” and subsequently 2’-O-methylated by Hen1, resulting in mature piRNAs. Although Trimmer has been characterized as a Mg²⁺ dependent 3’-5’ exonuclease, its identity remains unknown. We previously established a cell free system to monitor pre-piRNA 3’-end processing by using cell lysate from BmN4, a silkworm cell line expressing endogenous piRNAs. In BmN4 cells, most of the pre-piRNA trimming activity is detected in the insoluble fraction. Because of this insoluble nature, biochemical approaches to identify Trimmer have been extremely challenging.

(Results) Toward the identification of Trimmer, we have sought to solubilize the trimming activity. A cellular fractionation revealed that the trimming activity is enriched in mitochondrial fractions. Since the mitochondrial surface is thought to be a site of piRNA biogenesis, we focused on this fraction. We tried various detergents and finally succeeded in partial solubilization of the trimming activity from crude mitochondrial fraction. To isolate Trimmer, we next performed sucrose density gradient centrifugation. The trimming activity was detected relatively heavy fractions, suggesting that Trimmer forms a large complex. In JAJRNA meeting, we will report our current progress in biochemical identification of Trimmer.