Journal: Science advances

Article Title: The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

doi: 10.1126/sciadv.adf7001

Figure Lengend Snippet: Fig. 2. TAOK2β associates with the polyribosome complex, and its deficiency enhances general translation and protein synthesis. (A) Immunoblots of polysome fractions showing the presence or absence of TAOK2β across all polysome fractions isolated from Taok2+/+ (top) or Taok2−/− (bottom) mouse cortex. PABP1, RPL7a, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as positive and negative controls, respectively, to prove polysome preparation efficiency. (B) Polysome profiles from Taok2+/+ and Taok2−/− mouse cortices. (C) Quantification of polysome profiles in Taok2−/−, Taok2+/−, and Taok2+/+ mice cortices. Taok2+/+, n = 13; Taok2+/−, n = 8; Taok2−/−, n = 12; scanning electron microscopy (SEM) error bars, ordinary one-way analysis of variance (ANOVA) (P = 0.0251) followed by Dunnett’s multiple com- parisons test (*P < 0.05). (D) Immunostainings of acute cortical slices from the prefrontal cortex of Taok2+/+ and Taok2−/− mice analyzed by SUnSET assay. WT slices pre- treated with the translation inhibitor cycloheximide (WT + CHX) verifies the specificity of the puromycin antibody signal. (E) Immunoblot from acute cortical slices analyzed by SUnSET assay. (F) Quantification from (E). Taok2+/+, n = 5; Taok2+/−, n = 6; Taok2−/−, n = 5; SEM error bars, ordinary one-way ANOVA (P = 0.0167) followed by Dunnett’s multiple comparisons test (*P < 0.05). (G) Immunostainings of primary neurons (7DIV) analyzed by SUnSET assay. Cultured WT neurons were pretreated with CHX (WT + CHX). (H and I) Quantifications of immunofluorescent SUnSET assay of 7DIV neurons or 21DIV. 7DIV cells: Taok2+/+, n = 236 (five embryos); Taok2+/−, n = 113 (four embryos); Taok2−/−, n = 166 (four embryos). 21DIV cells: Taok2+/+ (three embryos), n = 81; Taok2+/−, n = 73 (two embryos); Taok2−/−, n = 79 (three embryos). SEM error bars, ordinary one-way ANOVA (P < 0.0001) followed by Dunnett’s multiple comparisons test (****P < 0.0001). Scale bars, 10 μm [(D) and (G)].

Article Snippet: The following antibodies were used in these studies for immunocytochemical or WB analysis: rabbit anti- Taok2β (Synaptic Systems, 395003; WB, 1:1000; Immunocytochemistry (ICC), 1:250; IP, 1:100); mouse anti- puromycin, clone 12D10 [Millipore, MABE343; WB, 1:10,000; immunohistochemistry (IHC); ICC, 1:5000]; chicken anti- MAP2 (Synaptic Systems, 188006; IHC, 1:500); guinea pig anti–β3- tubulin (Tuj1) (Synaptic Systems, 302304; ICC, 1:500); mouse anti–β- actin (Sigma- Aldrich, A5316; WB, 1:2000); rabbit anti–β- actin (13E5) (Cell Signaling Technology, 4970; WB, 1:5000); rabbit anti–Myc- tag (71D10) (Cell Signaling Technology, 2278; WB, 1:1000; ICC, 1:300); rabbit anti–immunoglobulin G (IgG) (Millipore, 03- 241; IP, 1:200); rabbit anti–phospho- eEF2 (Thr56) (Cell Signaling Technology, 2331; WB, 1:1000); rabbit anti–GSTtag (Cell Signaling Technology, 2625; WB, 1:1000); mouse anti– His- tag (27E8) (Cell Signaling Technology, 2366; WB, 1:1000); rabbit anti–His- tag (Cell Signaling Technology, 2365; WB, 1:1000); mouse anti- eEF2, clone 4B3- G7- H5 (Abcam, ab131202; WB, 1:2000); rabbit anti–phospho- eEF2K (Ser366) (Cell Signaling Technology, 3691; WB, 1:1000); rabbit anti- RPL7a (E109) (Cell Signaling Technology, 2415; WB, 1:1000); rabbit anti- PABP1 (Cell Signaling Technology, 4992; WB, 1:1,000); and mouse anti- eEF2K antibody (C- 12) (sc- 390710; WB, 1:200).

Techniques: Western Blot, Isolation, Electron Microscopy, Cell Culture

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: Phenotype of hda714 CRISPR/Cas9 mutants. ( A ) Designed HDA714 sgRNA sequence and position in the locus and the decoded mutations in two lines. ( B ) Phenotypes of hda714 mutant at germination, seedling and mature stages. Seeds were germinated in air or under submergence (sub) and photographed 3 and 6 days after germination (DAG). ( C ) Statistics of difference between wild type and hda714–1 germination rates ( n = 120) under air or submergence at 3 or 6 DAG, seed setting rates ( n = 9) at mature stage (Number of filled grains to total number of reproductive sites and expressed as percentage), and seedling heights (14 days, n = 15). For all the data, the means ± SEM of three independent biological replicates was shown. Significance of differences were analyzed using the two-tail Student's t -test ( E is *10 x ).

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques: CRISPR, Sequencing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: Relative protein lysine acetylation levels in hda705 , hda706 and hda714 compared with wild type. ( A ) Heatmap of relative lysine acetylation levels in two replicates of hda705, hda706 , hda714 and wild type. ( B – D ) Scattering plots of acetylation levels at individual Kac sites between wild type and the mutants. Significantly up and downregulated sites (>1.5-fold) in the mutants are indicated by red and green respectively. Up regulated (1–1.5-fold) and down regulated (1–1.5-fold) are indicated by light blue and tan respectively. ( E ) Numbers of Kac sites and proteins that were significantly up and downregulated (>1.5-fold) in hda705 , hda706 and hda714 compared with wild type.

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques:

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: HDA714 loss-of-function resulted in increases of lysine acetylation of translational proteins. Relative acetylation levels at individual Kac sites of ribosomal proteins ( A ) and translational initiation and elongation factors ( B ) in hda705, hda706 and hda714 compared with wild type. ( C ) Analysis of Kac levels of ribosomal proteins isolated from hda714–1 (two biological replicates) and wild type (two biological replicates) plants by immunoblotting using anti-LysAc antibody. Coomassie staining and immunoblotting with anti-RPS3 antibody were used as loading control. Two replicates are shown. ( D ) In vitro r-protein deacetylation assays. Ribosomal proteins isolated from wild type plants were incubated with E. coli -produced GST-HDA714 protein (with or without addition of sodium butyrate) followed by immunoblotting using anti-LysAc antibody. Coomassie staining was used as loading control. Two independent replicates are shown. ( E ) In vitro deacetylation assays of RPS3 and RPS6. E. coli -produced His-tagged RPS3 and RPS6 proteins were incubated with (+) or without (−) GST-tagged-HDA714 in the deacetylation butter and analyzed by western blots with anti-RPS3, RPS6, and anti-LysAc (acetylated lysine residues). Replicates are shown. ( F ) Effect of HDA714 loss- or gain-of-function on Kac on RPS3 and RPS6 in vivo in rice plants. RPS3 and RPS6 were immunoprecipitated from HDA714 mutants ( hda714–1 , hda714–2 ), overexpression (OE1, OE2) and wild type plants (WT1, WT2) by anti-RPS3 or anti-RPS6 and analyzed by immuno-blots using the indicated antibodies. Two replicates are shown. Band intensities in (E) and (F) were measured using ImageJ with those WT1 set at 1.

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques: Isolation, Western Blot, Staining, In Vitro, Incubation, Produced, In Vivo, Immunoprecipitation, Over Expression

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: In vivo protein interaction between HDA714 and ribosomal and IF proteins. ( A ) Tobacco leaves co-infiltrated with Agrobacterium containing 35S-driven split luciferase (LUC) constructs as indicated were photographed with a charge-coupled device camera. BiFC visualization showing that interaction between HDA714-Nluc and tested proteins (RPS16-cLUC, RPS3-cLUC, eIF2α-cLUC) in the N. benthamiana epidermal cells was observed. For the negative controls (nLUC + cLUC, HDA714-nLUC+cLUC, RPS6-cLUC+nLUC, RPS3-cLUC+nLUC, and eIF2α-cLUC+nLUC, HDA714-nLUC+WOX11-cLUC) no interaction was observed. eIF2α: eukaryotic translation initiation factor 2 alpha subunit. WOX11: WUSCHEL-RELATED HOMEOBOX11 (WOX11) as a negative control. ( B ) Coimmunoprecipitation assay for the interaction between HDA714 and RPS3, RPS6 or RPL7a in vivo . Total proteins from N. benthamiana leaves co-expressing RPS3-, RPS6- or RPL7a-FLAG and HDA714-GFP were immunoprecipitated using anti-GFP agarose. The agarose-bound proteins were eluted and detected by immunoblotting using anti-GFP or anti-FLAG antibodies. IP, immunoprecipitation. ( C ) Tests of in vivo interaction between HDA714 and RPS3 and RPS6 in rice plants. Proteins extracted from wild type rice plants were precipitated with IgG or anti-HDA714 and analyzed by immunoblots with anti-RPS3, anti-RPS6 and anti-HDA714. IP, immunoprecipitation.

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques: In Vivo, Luciferase, Construct, Negative Control, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: The hda714 mutation led to decreases of ribosomal proteins levels. ( A ) Box plots of abundance of ribosomal proteins detected in the hda714 and wild type proteomic data. ( B ) Box plots of acetylation levels of ribosomal proteins in hda714 and wild type plants. Horizontal lines in the boxes in (A) and (B) are medians; box limits indicate the 25th and 75th percentiles; whiskers extend to 5th and 95th percentiles. Significance of differences between wild type and the mutant indicated in the figures were analyzed using the two-tail Student's t -test ( E is *10 x ). ( C ) Scattering plots of ribosomal proteins and acetylation changes in hda714 versus wild type. Correlation coefficients are indicated. ( D ) Ribosomal proteins (K and K to Q or R) were fused with the GFP tag and expressed in N. benthamiana by agroinfiltration. The transfected N. benthamiana were treated with cycloheximide (CHX) plus or minus MG132, and harvested at the indicated time points. Total protein was analyzed by Western blots with anti-GFP. Immunoblotting results were quantified using ImageJ (v1.6.0_24). Values in R mutations at 0 h are set as 1.

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques: Mutagenesis, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: The hda714 mutation increased ribosomal binding frequency and characteristics of upregulated RBE mRNAs in the mutant. ( A ) Genome-wide metagene analysis of ribosomal footprinting density in gene coding region in wild type (blue) and hda714 (orange). RPKM: Reads Per Kilobase per Million mapped reads; TSS: translation start site; TES: translation end site. ( B ) Numbers of upregulated and downregulated (>2-fold, P < 0.05) genes detected by Ribo-seq in hda714 versus wild type. ( C ) Comparison of ribosome binding efficiency (RBE, Ribo-seq reads/RNA-seq reads) between wild type and hda714 . Numbers of genes (mRNA) with higher or lower (>2-fold, P < 0.05) RBE are indicated. ( D ) Expression level analysis of genes with upregulated RBE compared with genome-wide gene expression levels which was divided into 3 intervals: the highest 25% (TPM > 39.33), the lowest 25% (1 ≤ TPM ≤ 5.8) and the middle (25–75%) (5.18 < TPM ≤ 39.3). Genes with TPM <1 were considered as unexpressed and were not included here. TPM: transcripts per kilobase of exon model per million mapped reads. Significance of differences was analyzed using the two-tail Student's t -test. For boxplots, horizontal lines show medians; box limits indicate the 25th and 75th percentiles. ( E, F ) Comparison of coding region and 5′ UTR (untranslated region) lengths between the genes with upregulated RBE in hda714 and all rice genes. CDS lengths are negatively correlated with p values of RBE up genes (E). Significance of differences was analyzed using the two-tail Student's t -test ( E is *10 − x ). For boxplots, horizontal lines show medians; box limits indicate the 25th and 75th percentiles. ( G ) MEME motif of 5′UTR of genes with upregulated RBE in the mutant. E is *10 − x . ( H ) GO pathway enrichment analysis of RBE up genes ( N = 759).

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques: Mutagenesis, Binding Assay, Genome Wide, Footprinting, RNA Sequencing Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Histone deacetylases control lysine acetylation of ribosomal proteins in rice

doi: 10.1093/nar/gkab244

Figure Lengend Snippet: Ribosomal pause score analysis in hda714 and wild type. ( A ) Overall ribosome pause scores in wild type and hda714 . ( B ) Higher pause scores in hda714 upregulated RBE ( N = 759) genes. Two replicates are shown in (A) and (B). Significances of differences were analyzed using the two-tail Student t test. For boxplots, horizontal lines show medians; box limits indicate the 25th and 75th percentiles. E is *10 − x . ( C ) RiboToolkit analysis showed significant ribosome pausing at CAA (Gln) and AAA (Lys) codons in hda714 mutants in comparison to wild types (WT). Upper part: Codon occupancy changes between hda714 mutant and wild type. Lower part: Cumulative occupancy distribution of CAA (Q) and AAA (K) codons. P -values are from a one-sample Kolmogorov–Smirnov test.

Article Snippet: To confirm the interaction between HDA714 and ribosomal proteins in vivo , the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C.

Techniques: Mutagenesis

Journal: Advanced Science

Article Title: Pre‐rRNA Facilitates TopBP1‐Mediated DNA Double‐Strand Break Response

doi: 10.1002/advs.202206931

Figure Lengend Snippet: Pre‐rRNPs colocalize with TopBP1 at DSBs. A) Pre‐rRNA colocalizes with TopBP1 at DSBs. Following 10 Gy of IR treatment on HCT116 cells, RNA FISH with pre‐rRNA probes and IF with anti‐TopBP1 antibody were performed. B–D) Pre‐rRNA‐associated proteins colocalize with TopBP1 at DSBs. Following 10 Gy of IR, IF was performed with anti‐TopBP1 and anti‐RPL7A (B), or anti‐RPS3 (C), or anti‐FBL antibodies (D). E) Pre‐rRNPs localize at I‐SceI‐induced DSB. I‐SceI‐mediated DSB was induced by Dox and TA treatment in the HCT116 cells harboring a solo I‐SceI site. Then, ChIP assay with the indicated antibodies was performed. Data are represented as mean ± SD as indicated from three independent experiments. F) TopBP1 localizes at the unsynapsed region of the XY body. Meiotic spread was examined with anti‐TopBP1 antibody. Sycp3 was a surrogate marker of the unsynapsed axis of the XY body in pachytene cells. G) Pre‐rRNA localizes at the unsynapsed region. Pre‐rRNA was examined by RNA FISH with pre‐rRNA probes. H–J) Pre‐rRNA‐associated proteins localize at the unsynapsed region. Meiotic spreads were stained with anti‐Sycp3 and anti‐RPL7A (H), anti‐RPS3 (I), or anti‐FBL antibodies (J). The relative intensity of fluorescence signals is analyzed. Two‐tailed Student's t ‐test is used to determine statistical significance. *** p < 0.001, versus control groups. Scale bars, 10 µm.

Article Snippet: Detailed information of antibodies used in this study: anti‐Flag (Sigma, Rabbit, F7425), anti‐TopBP1 (Santa Cruz, Mouse, sc‐271043; BETHYL, Rabbit, A300‐111A), anti‐γH2AX (Novus, Rabbit, NB100‐384; CST, Rabbit, 9718), anti‐SCP3 (Santa Cruz, Mouse, sc‐74569; Novus, Rabbit, NB300‐232), anti‐rRNA (Novus, Mouse, NB100‐662), anti‐RPL7A (ABclonal, Rabbit, A13713), anti‐RPS3 (ABclonal, Rabbit, A2533), anti‐FBL (CST, Rabbit, 2639S; Santa cruz, Mouse, sc‐166001), IgG (Rabbit, CST, 2729), IgG (Mouse, CST, 3420), anti‐ATR (BETHYL, Rabbit, A300‐137A), anti‐Chk1 (Novus, Rabbit, NB100‐464), anti‐pATR (T1989) (GeneTex, Rabbit, GTX128145), anti‐pChk1 (Ser345) (CST, Rabbit, 2348), anti‐GAPDH (CST, Mouse, 97 166), anti‐pH3 (Ser10) (CST, Rabbit, 9701), anti‐Puromycin (Millipore, Mouse, MABE343), anti‐GAPDH (HUABIO, Rabbit, ET1601‐4), anti‐Histone H3 (CST, Rabbit, 4499S), anti‐ATM (GeneTex, Mouse, GTX70107), anti‐pATM (S1981) (ROCKLAND, Mouse, 200‐301‐400), anti‐pRPA (Novus, Rabbit, NBP1‐23017), anti‐Rad9 (Santa Cruz, Mouse, sc‐74464), anti‐RAD51 (abcam, Rabbit, ab63801).

Techniques: Marker, Staining, Fluorescence, Two Tailed Test