Fig. 2: amiRCWC15 reduces pri-miRNA accumulation through affecting transcription.
From: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis
a RT-qPCR analysis of pri-miRNA levels in Col-0 and amiRCWC15. pri-miRNA levels were normalized to those of ACTIN2 and compared with those in Col (set as 1). Error bars indicate SD from three biological replicates (n = 3) and data are presented as mean values +/- SD. Asterisks indicate significantly reduced expression compared to Col based on P-value (two-tailed unpaired t-test; P < 0.01):. MIR156a, 0.00161/0.000479; MIR167a, 0.00146/0.00022; MIR159a, 0.001097/0.000328; MIR167c, 0.002947/0.088748;MIR166a, 0.000175/0.00011;MIR172b, 0.000113/0.000107; MIR171c, 0.007848/0.002982;MIR164a, 0.002065/0.00199. b Histochemical staining of GUS in inflorescence of Col and amiRCWC15 harboring the pMIR167a::GUS transgene. Fifteen plants for each genotype were analyzed and a picture is shown (Scale bar: 0.5 cm). c GUS transcript levels in the indicated plants (the values in Col were set as 1) detected by RT-qPCR. Error bars indicate SD from three biological replicates and data are presented as mean values +/- SD. w/o Ab. indicates without antibodies. Asterisks indicate significantly reduced expression compared to Col based on P-value (two-tailed unpaired t-test; P < 0.01): 0.000135. d Pol II occupancy at MIR promoters in the indicated plants detected by ChIP followed by qPCR. The intergenic region between At2g17470 and At2G17460 (POL II C1) was used as a negative control. Error bars indicate SD from three biological replicates (n = 3) and data are presented as mean values +/- SD. Asterisks indicate significantly reduced occupancy based on P-value (two-tailed unpaired t-test; P < 0.05): MIR159a, 0.011092; MIR173, 0.043162; MIR168, 0.024155; MIR172b, 0.000269; MIR171a, 0.023305; MIR156a, 0.000283; MIR167a, 0.000271; POL II C1, 0.788731. e Co-IP between CWC15 and RPB2 in Col harboring the p35S::2HA-CWC15 transgene. Co-IP was performed using anti-HA. 2HA-CWC15 and RBP2 were detected by western blot. f The occupancy of CWC15 at MIR promoters in the indicated plants detected by ChIP followed by qPCR. The intergenic region between At2g17470 and At2G17460 (POL II C1) was used as a negative control. Error bars indicate SD from three biological replicates (n = 3) and data are presented as mean values +/- SD. Asterisks indicate significant occupancy based on P-value (two-tailed unpaired t-test; P < 0.05): MIR168a, 0.003198; MIR172b, 0.000133; MIR319b, 0.00024; MIR171a, 0.000321; MIR164a, 0.019332; MIR156a, 0.007154; MIR159a, 0.000671; MIR167a, 0.000435; MIR173, 0.001713, Source data are provided as a Source Data file.
