Fig. 6: Impaired Microprocessor assembly in PASH-1 WW domain mutants.

a Western blot analysis of wild-type and mutant PASH-1::GFP and wild-type mCherry::DRSH-1 from protein fractions captured with size exclusion chromatography. Masses are approximated based on size markers. The void fractions were determined based on protein elution volumes and confirmed with high molecular weight standards not run in parallel. Asterisks mark expected bands. b, c Western blot analysis of PASH-1::GFP and PASH-1[G179R]::GFP (b) or PASH-1[W180A]::GFP (c) co-IP’d with mCherry::DRSH-1. in, cell lysate input fraction; IP, co-IP fraction. Tubulin is shown as a loading control. Asterisks mark expected bands. The bar plots show the mean ratio of mCherry levels in IP fractions relative to GFP levels in IP fractions normalized to GFP levels in input fractions. Blue bar: mCherry::drsh-1; pash-1::GFP. Red bar: mCherry::drsh-1; pash-1[G179R]::GFP. Values are relative to the mCherry::drsh-1 pash-1::GFP control. Two-tailed, two-sample Student’s t-tests were used to calculate the p-values. Error bars are SD. n = 3 biological replicates. Samples derived from the same experiment and blots were processed in parallel. Blot images for one of 3 replicates are shown (Supplementary Fig. 6a–d). d Western blot analysis of PASH-1::GFP, PASH-1[G179R]::GFP, PASH-1[W180A]::GFP and mCherry::DRSH-1 co-IP’d with FLAG::PASH-1[149-266]. Tubulin is shown as a loading control. Asterisks mark expected bands. Blot images for one of 4 biological replicates from 2 independent experiments are shown (Supplementary Fig. 6e–f). e–g Mass spectrometry analysis of mCherry::DRSH-1 (e), PASH-1::GFP (f), and PASH-1[G179R]::GFP (g) complexes from protein co-IPs. Scatter plots display the average log₂ unique peptide counts in co-IPs from wild-type and the indicated transgenic strains. n = 4 biological replicates for each strain. Diagonal lines show 0-, 2-, and -2-fold enrichments. p-values were calculated based on label free quantification (LFQ) using a modified t-statistic (see Methods). The inset Venn diagram in (f) shows the overlap between mCherry::DRSH-1 and PASH-1::GFP interactors (p < 0.05). Source data are provided as a Source Data file.