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Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis - PubMed

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Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis

Yanxi Pei et al. Plant Physiol. 2007 Aug.

Abstract

Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis.

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Figures

Figure 1.
Figure 1.

Purification of a histone H4 methyltransferase from cauliflower. A, Purification scheme of the histone H4 methyltransferase. Numbers represent salt concentration (m

m

). BA, BC, and BD refer to buffer A, buffer C, and buffer D, respectively. Details are described in “Materials and Methods.” B, Silver-stained gel (top section) and histone methyltransferase activity against core histone (bottom section) of fractions derived from Superdex200 column. Proteins cofractionated with H4 methyltransferase activity are indicated by an arrowhead. Elution profile of Mr standard is indicated at the top. Numbers on the left indicate Mr standard of SDS-PAGE. In represents input. Numbers above the gel lanes indicate fraction number. C and D, Protein that cofractionated with histone H4 methyltransferase activity, PHRMT5, is nearly identical to AtPRMT5 by mass spectrometry analysis. Peptides derived from MSMS analysis are listed. Numbers represent amino acids number of AtPRMT5. Underlined amino acids are amino acids different between PHRMT5 in cauliflower and AtPRMT5 in Arabidopsis, which are also summarized in D. E, Mass spectrometry analysis of histone H4 methylated by PHRMT5 (top section). Histone H4 in a mock-performed histone methyltransferase activity assay where enzyme was omitted is used as a control (bottom section). Methylated peptides and corresponding unmodified peptide peaks are indicated. Ac-SGRGK, Ac-SGRmeGK, and Ac-SGRme2GK are unmethylated, R3 monomethylated, and R3 dimethylated peptides corresponding to amino acids 1 to 5 of histone H4. Peptide sequences are confirmed by tandem MS analysis (data not shown). [See online article for color version of this figure.]

Figure 2.
Figure 2.

Sequence alignment of PRMT5 homologs in Arabidopsis, human, mouse, and yeast. Conserved methyltransferase region I, posts I, II, III, and THW loop are labeled by lines.

Figure 3.
Figure 3.

Methyltransferase activity and specificity of recombinant AtPRMT5 in vitro. A, Recombinant proteins were incubated with indicated substrates in the presence of the methyl donor S-adenosyl-

l

-[methyl-3H]. Recombinant GST-AtPRMT5 methylates histone (a and b) and MBP (c and d). Coomassie blue-stained gel is shown on the left (a and c) and the autoradiograph of the gel is shown on the right (b and d). Positions of GST-AtPRMT5, histones, and MBP are indicated. B, Calf thymus histone methylated by GST-AtPRMT5 is probed with antibody against symmetric dimetylated histone H4R3 (Abcam, ab5823). GST is used as a negative control. The membrane is stripped and reprobed with antibody against histone H4 to serve as a loading control. C, Calf thymus histone methylated by PHRMT5 is probed with antibody against symmetric dimethyl histone H4R3 (Abcam, ab5823). Reaction performed in the absence of PHRMT5, which is indicated as “−” is used as a negative control. The membrane is stripped and reprobed with antibody against histone H4 to serve as a loading control. D, TLC analysis of H4R3 methylated by AtPRMT5. The positions of MMA, ADMA, and SDMA are visualized by ninhydrin staining (right section). Autoradiography of this plate shows that the radio activities comigrated with ninhydrin-stained MMA and SDMA but not ADMA (middle section). Autoradiography of GST alone used as negative control is shown on the left. Free 3H-labeled SAM is indicated. Separation starting site is indicated as Origin and solvent front is indicated as Front. [See online article for color version of this figure.]

Figure 4.
Figure 4.

AtPRMT5 gene structure, expression, and pleiotropic phenotypes in atprmt5-1 and atprmt5-2 mutants. A, AtPRMT5 gene structure and the T-DNA insertion sites of SALK lines. White boxes and gray boxes represent exons and 5′ or 3′ untranslated region, respectively. Lines mean introns. The T-DNA insertion sites in each mutant are indicated by triangles. In atprmt5-1 mutant, arrows represent two inverted T-DNA insertions. B, AtPRMT5 full-length but not the N-terminal transcript is absent in the atprmt5 mutants. Top sections: The total RNA from seedlings with four to five rosette leaves of atprmt5 mutants and wild-type Col is probed by RNA blot with full-length coding sequence of AtPRMT5. EF1α gene is used as a control for constitutive expression. Bottom sections: RT-PCR analyses were performed for the N-terminally expressed AtPRMT5 (AtPRMT5N) in wild-type Col and atprmt5-1 and atprmt5-2 mutants. Equal amounts of cDNAs were determined by RT-PCR at the Actin locus. C to F, Pleiotropic phenotypes of atprmt5 mutants. Growth retardation of young seedling leaves at 12 d (C) and primary roots at 9 d (D). E, Comparison of rosette leaves between wild-type Col and atprmt5 plants. F, atprmt5 mutants are late flowering. Plants shown here are 8 weeks old grown at 23°C under LD. aprmt5-1 × aprmt5-2 means F1 progeny from the crosses between aprmt5-1 and aprmt5-2.

Figure 5.
Figure 5.

Late flowering of atprmt5 is FLC dependent. A, Flowering time of atprmt5 mutants is assessed by total leaf number after plants stop producing new leaves under different conditions or treatments. LD + Ver (vernalization) treatment means plants grown at 4°C under SDs for 6 weeks before transferred to 23°C under LD. SD + GA indicates GA treatment under SDs. SD + ethanol is as a control. Bars represent

sd

. Under SD or SD + ethanol conditions, atprmt5 plants did not yet flower after producing 120 leaves and the experiment was terminated at this point. Bars represent

sd

. B, RNA-blot analysis of mRNA level of FLC and SOC1 in seedlings with four to five rosette leaves of wild-type Col and atprmt5 plants. EF1α is used as an internal control for constitutive expression. C, Phenotype (a) and flowering time (b) of atprmt5/flc-3 double mutant grown at 23°C under LD. Bars represent

sd

. D, Chromatin modifications at FLC locus. a, Diagram of FLC genomic DNA. A to G regions are amplified in ChIP assay (Bastow et al., 2004). b, Positive control ChIP assay. ChIP-PCRs of FLC-C are shown from immunoprecipitated by anti-AcH3 antibody using seedlings of Col and fld-4 mutants. The quantitative PCR results were shown in the right. c, Representative ChIP-PCR results are shown. Input is chromatin before immunoprecipitation that is diluted 25 times for PCR amplification. NoAb refers to precipitation performed without antibody. AcH3, H3K4 m2, and H4R3 SDM refer to the corresponding immunoprecipitations using antibodies against acetylated histone H3, dimethylated histone H3 at K4, and symmetric dimethylation at histone H4R3, respectively. FLC-A to FLC-G represent different regions of FLC chromatin (Bastow et al., 2004) and the diagram is shown at the top. TUB8 is used as an internal control. [See online article for color version of this figure.]

Figure 6.
Figure 6.

Expression levels of upstream regulators of FLC in atprmt5 plants. mRNA expression levels of FLC repressors (A) and FLC activators (B) are analyzed by real-time PCR. Relative amount of RNA levels normalized to UBQ (A) or Actin (B) is shown. Bars represent

sd

. For FCA, δ and γ forms are detected.

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