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1.
FIGURE 5.

FIGURE 5. From: Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs.

H2A.X-F is preferentially methylated, whereas symmetrically dimethylated H2A does not bind to nucleoplasmin. a, total stage II/III and stage VI oocyte extract and laid egg extract were immunoblotted as shown. b, nucleoplasmin was immunoprecipitated from crude egg extract (lane 2; lane 1 contains 0.5% of the input egg extract) and immunoblotted for nucleoplasmin, histones H3 and H2A, and R3me1 and R3me2s (Upstate). c, isolated histones from chaperone-bound complexes in the egg (lane 1) and from acid-extracted pronuclear chromatin (lane 2) were immunoblotted against R3me1 and R3me2s (Upstate); the bottom panel was Coomassie-stained. d, isolated egg histones were run on a Triton-acid urea gel and immunoblotted for H2A (left) and R3me1 (right; Upstate). The migration positions of H2A.X-F and H2A are noted, as shown previously (37).

Carola Wilczek, et al. J Biol Chem. 2011 December 9;286(49):42221-42231.
2.
FIGURE 2.

FIGURE 2. From: Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs.

Prmt5-Mep50 monomethylates and symmetrically dimethylates H2A at Arg-3. a, the peak activity fraction from the MonoQ was incubated with H2A and [3H]AdoMet and then subjected to mass spectrometry analysis. The MS/MS spectrum shown confirms that the activity resulted in H2A Arg-3 methylation; masses from y ions are underneath sequence, and b ions are above. Pr, propionyl. b, the peak activity fraction from the MonoQ (ePrmt5-Mep50) was incubated with H2A and AdoMet and then immunoblotted with antibodies directed against R3me1 and R3me2s (Upstate), demonstrating that Prmt5 methylates H2A on arginine. The figure is cropped for clarity from the same gel and exposure. c, egg-purified (e) Prmt5-Mep50 (in the peak MonoQ fraction from Fig. 1) was titrated into a methyltransferase reaction with a constant amount of H2A (1 μg), and the reaction products were immunoblotted with the Upstate antibodies against R3me1 (top), R3me2s (middle), and H2A (bottom).

Carola Wilczek, et al. J Biol Chem. 2011 December 9;286(49):42221-42231.
3.
FIGURE 6.

FIGURE 6. From: Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs.

A schematic model for the function of Prmt5-dependent methylation of histones H2A and H4 and the histone chaperone nucleoplasmin. During oogenesis, core histone proteins H2A, H2B, H3, and H4 are synthesized and stored by the storage chaperones Npm and N1. For clarity, in this figure we only show Npm and its known interacting partners H2A and H2B. The Prmt5-Mep50 complex is produced later in oogenesis, at which time we hypothesize that it methylates Npm and H2A/H2A.X-F and H4. Methylated Npm then acts upon fertilization to deposit histones, specifically monomethylated H2A.X-F and H4, in the growing chromatin. Specific effector proteins recognize the methylarginine in Npm and histones, during oogenesis and/or during early development. We hypothesize that these putative “readers” of methylarginine play a significant role in developmental regulation of gene expression and plasticity. Mep50 has six WD40 domains, illustrated as a single polypeptide (24).

Carola Wilczek, et al. J Biol Chem. 2011 December 9;286(49):42221-42231.
4.
FIGURE 1.

FIGURE 1. From: Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs.

Purification and identification of an abundant histone H2A and H4 arginine 3 methyltransferase in Xenopus laevis egg extract as a complex of Prmt5 and Mep50. a, Xenopus egg extract was incubated with recombinant histones H2A, H2B, H3, and H4 and with the methyl donor [3H]AdoMet ([3H]SAM). Shown are a Coomassie-stained SDS gel (bottom) and a fluorogram (top). b, recombinant H2A was incubated with egg extract and [3H]AdoMet in the presence or absence of 50 μm AMI-1. Bottom, Coomassie-stained SDS gel; top, corresponding fluorogram. c, chromatography scheme for the purification of the H2A methyltransferase activity. d, full-length histone H2A methyltransferase activity across the fractions of the MonoQ column (final column in the purification) performed as a P81 filter binding assay; the HMTase activity histogram showed peak activity in fractions 32–34. e, silver-stained gel of the peak activity fractions. f, Coomassie-stained SDS gel of the pooled peak activity fractions 32–34. Stained bands were identified by mass spectrometry (supplemental Fig. S1), and the location of the arginine methyltransferase Prmt5 and its known cofactor Mep50 are shown with arrows on the gel and in the silver-stained gel. g, Coomassie-stained SDS gel of recombinant Prmt5 and Mep50 produced in baculovirus-infected Sf9 cells. h, methyltransferase assay demonstrating that the recombinant Prmt5-Mep50 complex methylates recombinant H2A (fluorogram, top); lane 1 did not contain enzyme, whereas lane 2 did contain enzyme, and both lanes contained H2A.

Carola Wilczek, et al. J Biol Chem. 2011 December 9;286(49):42221-42231.
5.
FIGURE 4.

FIGURE 4. From: Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs.

Prmt5 methylates the histone chaperone nucleoplasmin on a conserved motif in its C-terminal tail. a, egg extract incubated with or without 1 μg of recombinant Npm and 1 mm AMI-1 in the presence of [3H]AdoMet. Reactions were run on an SDS gel, stained with Coomassie Blue, and exposed for fluorography. rNpm, recombinant nucleoplasmin. b, recombinant Prmt5-Mep50 complex (left) or recombinant Prmt1 was incubated in with H2A, nucleoplasmin, or both and with [3H]AdoMet; the reactions were Coomassie-stained (bottom) and exposed for fluorography (top). c, purified recombinant Npm, oocyte Npm, and egg Npm were immunoblotted for Npm, monomethylarginine, and symmetric dimethylarginine; the Upstate antibodies that recognize Arg-3 methylation were used for Npm. The Coomassie-stained SDS gel of the purified proteins is shown at the bottom. d, egg extract (immunoprecipitation (IP) input), protein A resin without antibody, resin coupled to R3me1 or R3me2 (both from Proteintech), or Prmt5 antibody incubated in egg extract were blotted for Npm. e, the first 10 amino acids of histones H2A, H2A.X-F, and H4 and the C-terminal amino acids of nucleoplasmin are shown. The known (histones) and putative (nucleoplasmin) Prmt5-targeted arginines are boxed. f, recombinant wild type and mutant (1–185 C-terminal truncation, R187A, R189A, and R187A/R195A) nucleoplasmin were incubated with 0.2 μg of recombinant Prmt5-Mep50 and [3H]AdoMet; the reactions were Coomassie-stained (bottom) and exposed for fluorography (top). g, recombinant wild type or mutant R187A/R189A nucleoplasmin were incubated with all four core histones, H2A-H2B dimers, or histone octamers and with 0.2 μg of recombinant Prmt5-Mep50 and [3H]AdoMet; the reactions were Coomassie-stained (bottom) and exposed for fluorography (top). h, recombinant Prmt5-Mep50 complex and [3H]AdoMet were incubated alone or with H2A, H4, or H2A-H2B dimers or H2A-H2B-H3-H4 histone octamers and also incubated with recombinant nucleoplasmin (first column of panels), oocyte nucleoplasmin (second column of panels), or egg nucleoplasmin (third column of panels). The reactions were Coomassie-stained (bottom) and exposed for fluorography (top).

Carola Wilczek, et al. J Biol Chem. 2011 December 9;286(49):42221-42231.
6.
FIGURE 3.

FIGURE 3. From: Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs.

Prmt5 exists only in complex with Mep50 in vivo and is found in the nucleus and on chromatin. a, total clarified egg extract was applied to a Superose 6 sizing column, and every other fraction was immunoblotted for Prmt5 and Mep50. Elution positions of 670 and 158 kDa mass markers are shown above. b, endogenous co-immunoprecipitation of Prmt5 with α-Mep50 in egg extract. c, 0.5 μl of clarified egg extract and 0.5 μl of NPE were immunoblotted for Prmt5 and PCNA (a nuclear marker). A small proportion of Prmt5 was found in the nuclear extract. d, sperm pronuclei were assembled in egg extract and collected immediately after incubation (1 min; first lane) or after 45 min (second lane). Chromatin was isolated through a sucrose cushion and immunoblotted for Prmt5, H2A, H3, and Mcm7. e, rPrmt5-Mep50, total egg extract, XL2 whole cell extract, and A6 whole cell extract were immunoblotted for Prmt5, Mep50, and PCNA. The bottom panel shows the total protein content in each lane by Direct Blue 71 staining of the membrane. The protein contents for the egg and whole cell extracts were normalized by a Bradford assay, with an identical total mass of protein loaded in each lane. r, recombinant. f, purified histones from Xenopus eggs, XL2 cells, and A6 cells were immunoblotted for H2A (as control) and R3me1 (Proteintech). The antibodies recognize both H2A and H2A.X-F in the egg extract; for the R3me1 antibody, we refer to both as H2A/H2A.X-F because we cannot distinguish between H2A and its variant in this case. Equal masses of histones were loaded in each lane as shown by Coomassie staining (bottom). IP, immunoprecipitation.

Carola Wilczek, et al. J Biol Chem. 2011 December 9;286(49):42221-42231.

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