Fig. 4: HRDE-1-HRDE-2 interaction is modulated by small RNA binding.

a Small RNA binding assay following immunoprecipitation of HRDE-1, HRDE-1(HK-AA), and HRDE-1 in a mut-2 mutant. The assay utilized different PCR cycles (10X, 12X, 14X, 16X) to assess the amount of small RNAs immunoprecipitating with HRDE-1. Arrows point to the band of the correct size for small RNA plus ligated adapters. Detailed protocol for the small RNA binding assay can be found in the Methods. b Quantification of HRDE-1-bound small RNAs from the small RNA binding assay. Average pixel intensities from two biological replicates are normalized to wild-type 10X PCR cycles. c. Western blot following immunoprecipitation of HRDE-1 in wild-type, mut-2 mutants, and hrde-1(HK-AA) mutants, where HRDE-1 was tagged with GFP::3xFLAG and HRDE-2 was tagged with 2xTy1::GFP. The HRDE-2::2xTy1::GFP strain was used as a negative control. Anti-FLAG conjugated beads were used to immunoprecipitate GFP::3xFLAG::HRDE-1 and anti-FLAG, Anti-Ty1, and anti-Actin antibodies were used to detect HRDE-1, HRDE-2, and Actin. Band intensities were quantified using ImageJ and normalized to wild-type (lane 2) to determine how the HRDE-1-HRDE-2 interaction is affected by the mut-2 and hrde-1(HK-AA) mutants. In this replicate, mut-2 and hrde-1(HK-AA) mutants lead to 2.18-fold and 3.3-fold increase in HRDE-1-HRDE-2 interaction, respectively. This co-immunoprecipitation and western blot have been repeated three times with similar results. Additional replicates are shown in Supplementary Fig. 4. Source data are provided as a Source Data file.