Sytnikova YA et al. (2011), Gadd45a is an RNA binding protein and is locali...

XB-ART-43671

PLoS One 2011 Jan 07;61:e14500. doi: 10.1371/journal.pone.0014500.

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Gadd45a is an RNA binding protein and is localized in nuclear speckles.

Sytnikova YA , Kubarenko AV , Schäfer A , Weber AN , Niehrs C .

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The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids. Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function. The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle.

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Species referenced: Xenopus
Genes referenced: alb gadd45a hnrnpa1 hnrnpc mt-tr smarca4 srsf2 trna

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	Figure 1. Gadd45a binds RNA in vitro.A, RNA filter binding assay using the indicated proteins and 32P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; M-MLV RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (nâ€Š=â€Š3). A representative experiment out of three is shown.
	Figure 2. Gadd45a is part of a large RNase sensitive complex.AâC Sedimentation analysis of RKO nuclear extracts in a linear 8â40% (top-bottom) sucrose gradient. Fractions were analyzed by Western blot for Gadd45a, hnRNP A1 and p68 (control RNA binding proteins) and Brg1 (negative control). Prior to sedimentation nuclear extracts were left untreated (A), DNAse treated (B), or RNAse treated (C). b, resuspended micro-pellet of tube. Representative experiment out of three is shown.
	Figure 3. Gadd45a localization in nuclear speckles is RNase sensitive.A, Scheme of detergent extraction. B, RKO cells expressing EGFP-Gadd45a were subjected to detergent extraction with or without RNaseA treatment followed be Western blot analysis of the indicated proteins. A representative experiment out of three performed is shown. C, D Immunofluorescence confocal microscopy of detergent-extracted RKO cells. Cells were transfected with N-EGFP-Gadd45a and stained with antibodies against SC35 (C) and p68 (D). E, cells were treated as in B, but subjected to RNase treatment after extraction and before fixation. F, Statistical analysis for localization of EGFP-Gadd45a and SC-35 in nuclear speckles in cells with and without RNaseA treatment (nâ€Š=â€Š35 cells; nâ€Š=â€Š3 experiments; a representative experiment is shown). G, H Immunofluorescence confocal microscopy of endogenous Gadd45a in UV irradiated detergent-extracted HEK293T cells. Cells were stained with antibodies against Gadd45a and SC35. In (H), cells were subjected to RNase treatment after extraction and before fixation. I, Statistical analysis for localization of UV inducible Gadd45a in nuclear speckles in HEK293T cells with and without RNaseA treatment (nâ€Š=â€Š50 cells).
	Figure 4. Gadd45a modeling suggests domains of RNA binding.A, Sequence alignment of L7Ae family proteins: human hsp15.5 kDa protein, yeast ribosomal scL30e protein, Haloarcula marismortui ribosomal hmL7Ae protein, yeast spliceosomal protein scSnu13p protein, human hsSBP2_RBD (RNA-binding domain), human hsGadd45g and Xenopus tropicalis xtGadd45a, including secondary structure elements (above) and sequence conservation (below). Light and dark blue letters indicate backbone- and side chain RNA interacting residues from patch 1. Light and dark green letters indicate backbone and hydrophobic side RNA interacting residues from patch 2 (see text for details). Residues targeted by mutagenesis are marked. BâE, Comparison of the crystal structures of human hsp15.5 kDa protein (B), yeast ribosomal scL30e protein (C) and Haloarcula marismortui ribosomal hmL7Ae protein (D), and the homology model of Xenopus tropicalis xtGadd45a (E). Residue coloring as above. The red area denotes the ultra-conserved Gly residue (RNA guanine G-binding region) important for specific RNA binding and DNA demethylation.
	Figure 5. RNA binding and DNA demethylation in Gadd45a point mutants.A, general characteristics of Gadd45a point substitutions. B, SDS-PAGE analysis of His-tagged Gadd45a wild type and point mutant proteins produced and purified from E.coli. C, filter binding assay of Gadd45a wild type and point mutant proteins using multiple cloning site (MCS) 32P-RNA. D, RNA filter binding assays using 32P-labeled MCS RNA were performed with wild type or point mutant Gadd45a proteins in the presence of the indicated unlabeled competitor RNAs. Data are pooled from seven independent experiments. E, F DNA demethylation assays. E, Luciferase reporter assays of HEK293T cells transiently transfected with an M. SssI in vitro methylated SV40-luciferase reporter and the indicated constructs. Error bars, s.e.m. (nâ=â3). F, Methylation sensitive Southern blot of HpaII in vitro methylated pOctTK reporter recovered from HEK293T cells cotransfected with Xenopus Gadd45a wild type and mutants.
	Figure 6. G39A substitution weakens Gadd45a association with nuclear speckles.A,B IF microscopy comparison of nuclear pattern after detergent extraction of EGFP-Gadd45a wild type (A) and EGFP-G39A mutant (B). Experiments were done essentially as in Figure 3A. C, Statistical analysis of immunofluorescence patterns as in Figure 3F. D, Western blot analysis of RKO cells expressing EGFP-Gadd45a wild type (wt) or EGFP-G39A mutant harvested without or after detergent extraction. Scale bar, 4 Âµm.

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