Fig. 4: CWC15 is required for miRNA maturation.
From: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis
a, b Schematic diagram of the MIR162b (a) and pre-miR162b (b) used in vitro processing assay. c and d miR162b produced from MIR162b and pre-miR162b in proteins extracts from inflorescences of amiRCWC15 or Col. The reactions were stopped at various time points as indicated in the picture. e and f Quantification of miR162b production in amiRCWC15 compared to that in Col. Quantification analysis was performed at 80 min. The radioactive signal of miR162b were normalized to input and compared with that of Col-0. The amount of miR162b produced in Col was set as 1. Error bars indicate SD from two biological replicates (n = 2) and data are presented as mean values +/- SD. ***P < 0.001 (P = 2.66793E-05 in a; P = 4.18939E-05 in b; two-tailed unpaired t-test). g and h The association of HYL1 with pri-miRNAs in amiRCWC15 relative to Col detected by RNA immunoprecipitation. IP was performed with anti-HYL1 antibodies. HYL1 was detected by Western blot. Pri-miRNAs associated with HYL1 were examined by RT-qPCR (h) and normalized to the input. UBQ5 serves as a negative control. w/o Ab. indicates without antibodies. Error bars indicate SD from two biological replicates (n = 2) and data are presented as mean values +/− SD. Asterisks indicate significantly reduced association with pri-miRNAs compared to Col based on P-values (two-tailed unpaired t-test; P < 0.05): MIR172b, 0.002867; MIR167a, 0.000157; MIR156b, 0.020845; MIR156a, 0.027099; MIR159a, 0.000751; MIR173, 0.030127; UBQ5, 0.898117. Source data are provided as a Source Data file.
