Fig. 2: The CYC-YPMF motif is essential for copurification of Pol IV with its recruitment factors.
a Experimental setup to evaluate the effect of mutations in the CYC-YPMF motif on the co-immunoprecipitation of proteins associated with the Pol IV complex (pink) using the NRPD1-3xF variants indicated below. The wild-type AtNRPD1 CYC-YPMF amino acids are indicated in purple and the amino acids altered in the other variant lines are outlined in red. These constructs were transformed into the nrpd1-3 null mutant and experiments were conducted in the T2 generation. b Balloon plots showing the results from co-immunoprecipitation and mass spectrometry (IP-MS) experiments using flower extracts from three independent lines, each with two technical replicates, expressing the NRPD1-3xFWT, NRPD1-3xFAAA-YPMF, or NRPD1-3xFCYC-AAAA variants. The plot shows protein spectral counts for Pol IV-RDR2 subunits (NRPD1 to NRPD12, RDR2) and other interactors (CLSY1-4 and SHH1) in each dataset. The spectral count is indicated by the log10-transformed balloon area. Purple shaded balloons represent subunits where comparisons between the NRPD1-3xFWT data and either the NRPD1-3xFAAA-YPMF or the NRPD1-3xFCYC-AAAA data pass fold change (FC) and adjusted (adj.) p-value cut-offs, indicating their significance. By contrast, comparisons with |log2FC | <1 or adj. p-value > 0.05 are shown in gray. In the NRPD1-3xFWT data, the left half-balloon colors are adj. p-values for comparison to the NRPD1-3xFAAA-YPMF data, and the right half-balloon colors are adj. p-values for comparison to the NRPD1-3xFCYC-AAAA data. c, d Volcano plots showing the enrichment or depletion of proteins from six independent IP-MS experiments comparing the NRPD1-3xFWT lines with either the NRPD1-3xFAAA-YPMF or the NRPD1-3xFCYC-AAAA lines. The red hashed lines demarcate a log2FC of ±1 and an adj. p-value of 0.05. Purple dots indicate Pol IV recruitment factors (CLSY1-4 and SHH1), green dots the Pol IV core subunits, and a yellow dot represents RDR2. The adj. p-values derive from a quasi-likelihood negative binomial generalized log-linear model in IPinquiry4. e Anti-FLAG western blot detecting NRPD1 and CLSY3 proteins using the genotypes indicated above each lane from either input or streptavidin IP samples. Unless marked, all samples were loaded at 1x. The bands corresponding to NRPD1 as well as both full length CLSY3 (CLSY3FL; Black) and several CLSY3 degradation products (CLSY3deg; Gray) are indicated on the right. f Western blots detecting SHH1 and NRPD1 proteins in an Anti-HA IP using flowers from F1 plants of the self-fertilized SHH1-3xHA line, or flowers from this line crossed to the NRPD1-3xF variants indicated. g Western blots detecting CLSY1, NRPD1 and NRPD2 in an Anti-HA IP using flowers from 3xHA-CLSY1 plants supertransformed with the NRPD1-3xF variants indicated. h Western blots detecting RDR2 and NRPD1 proteins in an Anti-GFP IP using flowers from F1 plants of the self-fertilized RDR2-eGFP line, or flowers from this line crossed to the indicated NRPD1-3xF variants. The western analysis in panel e was performed two times, while the analyses in panels f, g and h were each performed once. Source data are provided as a Source Data file.
