Tethered Function Assay for Analyzing PIWI-piRNA-Mediated Co-transcriptional Silencing.

PIWI-interacting RNAs (piRNAs) form complexes with a series of PIWI proteins to silence their target transposons. In the nucleus, piRNAs associated with Drosophila Piwi protein induce co-transcriptional silencing. To understand this regulation, reporter-based forced tethering of Piwi-associated proteins has been utilized as a powerful tool. Here, we describe the use of luciferase reporter assays combined with forced tethering of Piwi-interacting proteins for investigation of the molecular mechanism of Piwi-directed co-transcriptional silencing in Drosophila. Here, ovarian somatic cells (OSCs) are used as an example for the construction of this reporter system, but this methodology can also be applied to in vivo analysis using Drosophila ovaries.