Fig. 2: Requirement for PASH-1’s WW domain in pri-miRNA recognition or processing.

a Representative images of control and 38a mutants containing the pri-miR-58 sensor. Scale bars = 0.1 mm. At least ten embryos per condition were imaged. b Quantification of mCherry levels in non-mutant control and 38a mutants by Western blot. One of 3 representative blot images is shown (see Source Data file). Tubulin was used for normalization. Error bars are SD. n = 3 biological replicates. A two-tailed, two-sample Student’s t-test was used for statistical analysis. c Location of the 38a mutation on pash-1 DNA and protein. dsRBDs, double-stranded RNA-binding domains. d WW domain sequence alignment. e AlphaFold3-predicted structure of the C. elegans Microprocessor highlighting the WW domain (residues 174–207) and dimerization region (residues 148-266). Human X-ray diffraction-based structure of the DGCR8 WW domain (PDB: 3LE4) is shown for comparison. f, g Relative endogenous pri-miR-58 levels in wild-type and pash-1(ram33[G179R]) mutants grown at 20 °C or 25 °C as measured by TaqMan qRT-PCR and normalized to act-1 (f) or unnormalized (g). Error bars are SD. n = 4 biological replicates. Two-tailed, two-sample Student’s t-tests were used for statistical analysis. A Bonferroni correction for three comparisons was applied in (g). h, i Relative mature miR-58 levels in wild-type and pash-1(ram33[G179R]) mutants grown at 20 °C or 25 °C as measured by TaqMan qRT-PCR and normalized to 21UR-1 (h) or unnormalized (i). Error bars are SD. n = 4 biological replicates. Two-tailed, two-sample Student’s t-tests were used for statistical analysis. A Bonferroni correction for three comparisons was applied in (i). j Numbers of progeny produced by animals grown at 20 °C. Error bars are SD. n = 10 (wild-type) or 11 (pash-1(ram33[G179R]) and pash-1(syb4327[W180A])) individuals. Two-tailed Mann-Whitney U tests were used for statistical analysis. k, l Images of wild-type and pash-1(ram33[G179R]) (k) or pash-1(syb4327[W180A]) (l) animals grown at 20 °C or 25 °C as indicated. Bar plots show percentages of animals with the indicated phenotypes (n = 100 animals per strain). Images approximate phenotypes scored. Two (k) or 1 (l) independent experiments were done. Scale bar = 0.3 mm. Source data are provided as a Source Data file.