a300 311a  (Bethyl)

Journal: bioRxiv

Article Title: Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

doi: 10.1101/2024.05.08.592654

Figure Lengend Snippet: A , Strategy for Inducible Disruption of Phosphatase Targeting (InDisruPT). Structure of a genomic transgene is depicted, before and after FLP-mediated recombination at FRT sites flanking a 3’ exon encoding the C-terminus of PNUTS. Shown are the wildtype 3’ exon, with GFP coding sequence in green, and non-PP1 binding variant, with mCherry in red. Recombination results in production of extragenic wildtype circular DNA, which is not maintained, and reconstruction of the PNUTS transcription unit carrying the alternative 3’ exon, which harbours the W726A mutation. B , Western blots showing that inducible disruption of PP1 binding to PNUTS results in Cdk11 Ser 712 phosphorylation in vivo . Adult flies carrying InDisrupt PNUTS transgenes, were induced with hsFLP and blotted with GFP, RFP, Cdk11-pSer 712, total Cdk11, and Actin antibodies either 0, 24, or 48hr after induction. C , Confocal images of early-stage egg chamber showing switching of PNUTS WT GFP: PNUTS W726A mCh specifically in germline cells using ovoFLP, and the effect on Cdk11-pSer712 staining. Switching is incomplete in some cells, as shown with a high GFP:RFP ratio. Scale bars, 50 µm. Line indicates position of line scan taken through the image for quantitation. D , Plots of line scan signal intensities for pCdk11, RFP, GFP and DNA (TO-PRO-3 dye) staining, showing effect of switching PNUTS WT GFP to PNUTS W726A mCh on Cdk11-pSer712 in nurse cell nuclei.

Article Snippet: Genomic transgenes : A 6kb genomic cdk11 construct was made by cloning a synthetic DNA fragment encompassing the cdk11 transcription unit (GeneArt, Thermofisher), -705bp of the ATG to +359bp of the stop codon, into the Bam HI/ Not I sites of the fly transformation vector pW8-attB.

Techniques: Disruption, Sequencing, Binding Assay, Variant Assay, Mutagenesis, Western Blot, Phospho-proteomics, In Vivo, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

doi: 10.1101/2024.05.08.592654

Figure Lengend Snippet: A , Multiple sequence alignment of Cdk11 proteins showing evolutionary conserved features, including DFG motif (boxed), Serine DFG+7 (yellow highlight) and the activating Threonine at DFG+13 (magenta highlight). As, Anopheles sinensis (mosquito); Dr, Danio rerio (zebrafish); Dm, Drosophila melanogaster (fly); Ec, Equus caballus (horse); Fd, Fukomys damarensis (rat): Gallus gallus (chicken); Hs, Homo sapiens (human); Mf, Marmota flaviventris (marmot); Mm, Mus musculus (mouse); Og, Otolemur garnetti i (Galago); Sp, Schizosaccharomyces pombe (fission yeast). B , Effect of Cdk11 DFG+7 amino-acid mutations on Cdk11/Cyclin L activity towards recombinant RNAPII-CTD. RNAPII-CTD was incubated with or without recombinant WT or mutant variants of Cdk11/Cyclin L, before immunoblotting with RNAPII-CTD antibodies specific for pSer2, 5, 7 or pThr4 or total RNAPII-CTD. Cdk11 WT /Cyclin L and Cdk11 S712A /Cyclin L showed activity towards RNAPII-CTD Ser5, whereas Cdk11 S712E /Cyclin L and Cdk11 KD /Cyclin L showed reduced and no activity, respectively. C , Quantification of pSer5 RNAPII-CTD band intensities, normalised to total RNAPII-CTD (n=3). Data shown are mean ±SEM (n=3). **, p=<0.01, one-way Anova. D , Time-dependent phosphorylation of CTD peptide 5-FAM-(YSPTSPS) 3 -CONH 2 , by CDK7/Cyclin H (∼0.5μg, positive control) and Cdk11/Cyclin L1 variants (∼1μg enzyme). Mean ±SD, n=2.

Article Snippet: Genomic transgenes : A 6kb genomic cdk11 construct was made by cloning a synthetic DNA fragment encompassing the cdk11 transcription unit (GeneArt, Thermofisher), -705bp of the ATG to +359bp of the stop codon, into the Bam HI/ Not I sites of the fly transformation vector pW8-attB.

Techniques: Sequencing, Activity Assay, Recombinant, Incubation, Mutagenesis, Western Blot, Phospho-proteomics, Positive Control

Journal: bioRxiv

Article Title: Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

doi: 10.1101/2024.05.08.592654

Figure Lengend Snippet: A , Genomic region of Drosophila cdk11 showing exon-intron structure for cdk11 and spatial relationship to flanking genes on chromosome 3L. Orange shading represents coding regions, grey boxes represent untranslated regions. cdk11 P contains an 3xPS>DsRed marked PBac element inserted into the coding sequence of cdk11 by CRISPR-Cas9 mediated gene targeting. Removal of PBac generated seamless mutation of S538/712 ( cdk11 SE ) due to site-directed mutations present in the homology arm. Also shown is the position of wild type genomic fragment containing the cdk11 transcription unit, which were used to make transgenic lines (cdk11 wt and site-directed mutants cdk11 SA and cdk11 SE ). B , Graph showing percentage of hetero- and homozygous cdk11 P animals surviving over time (days after egg laying). More than 90% of cdk11 P died by 72 hr. C , Graph showing ability of cdk11 genomic transgenes to rescue lethality caused by cdk11 loss-of-function. Shown are the proportion of offspring from genetic crosses that were either heterozygous (orange data points) or homozygous (blue data points) for cdk11 P . Mean ±SEM of three biological repeats is shown. The expected number homozygous cdk11 P animals upon complete rescue by cdk11 transgenes (33%) is indicated with a red dashed line. Both cdk11 wt and non-phosphorylatable cdk11 SA transgenes fully rescued cdk11 P lethality, whereas only partial rescue was observed with phospho-mimetic transgenic cdk11 SE . *, p<0.05, ** p<0.001 one-way Anova. D , Western blots of protein lysates from homozygous or heterozygous cdk11 P and cdk11 SE mutant larvae, showing loss of Cdk11 protein in cdk11 P extracts. Actin immunoblots show loading control. pCDK11 blots show lack of signal in either cdk11 P and cdk11 SE homozygous extracts, further confirming specificity of this antibody for phospho-Cdk11. E , Plot showing survival of cdk11 SE homozygous males (median survival 39.4 days) compared to w 1118 control (median survival 58.4 days).

Article Snippet: Genomic transgenes : A 6kb genomic cdk11 construct was made by cloning a synthetic DNA fragment encompassing the cdk11 transcription unit (GeneArt, Thermofisher), -705bp of the ATG to +359bp of the stop codon, into the Bam HI/ Not I sites of the fly transformation vector pW8-attB.

Techniques: Sequencing, CRISPR, Generated, Mutagenesis, Transgenic Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

doi: 10.1101/2024.05.08.592654

Figure Lengend Snippet: Total RNA from cdk11 P and cdk11 P homozygous larvae and developmentally-matched controls were analysed by RNA-Seq. A , Unsupervised hierarchical clustering of samples based on their DESeq2 normalized gene-level counts shows the degree of similarity between samples. The heat map displays inter-sample Euclidean distances, where darker blue colours indicate closer similarity. B , Venn diagram showing the overlap between the genes significantly differentially expressed in cdk11 P or cdk11 P (more than 1.5 fold increased or decreased compared to controls, (p adj<=0.05) showing common and divergent responses. Numbers of genes in each intersect are indicated, along with the percentage of genes represented from each genotype. C , Gene Ontology enrichment amongst significantly overexpressed or underexpressed genes in cdk11 P or cdk11 P generated using Metascape. The scale bar indicates the negative log(10) p-values for enrichment. Only significant categories are shown, and grey coloured cells indicate non-significance. Metabolic process and Response to stimuli are commonly enriched categories. D , Plots of log(2) fold change in expression of intronic snoRNAs (bottom) and their respective host genes (top) in cdk11 P (blue data points) or cdk11 SE (red data points) larvae.

Article Snippet: Genomic transgenes : A 6kb genomic cdk11 construct was made by cloning a synthetic DNA fragment encompassing the cdk11 transcription unit (GeneArt, Thermofisher), -705bp of the ATG to +359bp of the stop codon, into the Bam HI/ Not I sites of the fly transformation vector pW8-attB.

Techniques: RNA Sequencing, Generated, Expressing

Journal: bioRxiv

Article Title: Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

doi: 10.1101/2024.05.08.592654

Figure Lengend Snippet: A,B , Percent spliced in index (PSI) plots showing significant differences ( padj <=0.05) in splicing events identified between the control and cdk11 mutant strains. Colour key indicates different categories of splicing events that were examined using NxtIRF: intron retention (IR), mutually exclusive exons (MXE), skipped exons (SE), alternate first exon (AFE), alternate last exon (ALE), alternate 5’-splice site (A5SS) and alternate 3’-splice site (A3SS). Intron retention events (IR, cyan colour) are increased in cdk11 P compared to its matched control ( A ). In contrast, there were fewer intron retention events in cdk11 SE animals compared to the control ( B ). C , Plot of mean difference in RNAPII elongation speed in cdk11 P and cdk11 SE compared to the respective controls. The log-2 fold change in mean coverage between mutant and control samples is shown, oriented so that negative values represent more negative slopes and slower estimated transcription in the mutants. D, cdk11 P animals show an increase in aberrant intergenic transcription, as measured by the percentage of RNA-Seq reads mapping to intergenic bases (% intergenic bases, PICARD). T-test p-values are indicated above each mutant-control group comparison (n=3). E-G , cdk11 mutants differentially modulate transcriptional read-in and readthrough compared to control animals. E , Boxplot showing the median log(2) ratio of read-in expression (1-15 kb upstream of genes) to gene expression. F , Boxplot showing the median log(2) ratio of read-through expression (downstream, 4-15 kb in length) to gene expression. G , Boxplot of median downstream of gene expression (FPKM). In each panel, the results of T-tests between matched mutant-control pair groups are shown (n=3 per group).

Article Snippet: Genomic transgenes : A 6kb genomic cdk11 construct was made by cloning a synthetic DNA fragment encompassing the cdk11 transcription unit (GeneArt, Thermofisher), -705bp of the ATG to +359bp of the stop codon, into the Bam HI/ Not I sites of the fly transformation vector pW8-attB.

Techniques: Control, Mutagenesis, RNA Sequencing, Comparison, Expressing, Gene Expression

Journal: Nature

Article Title: CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

doi: 10.1038/s41586-022-05204-z

Figure Lengend Snippet: Fig. 2 | CDK11 kinase activity is needed for efficient pre-mRNA splicing. a, Differentially expressed genes from nuclear RNA-seq in HCT116 cells treated with 30 nM OTS964 for 4 h. Downregulated and upregulated genes are shown in red and blue, respectively. Padj, adjusted P. b, IGV genome browser view of WEE1 from RNA-seq. c, The ratio of spliced reads over total unspliced and spliced reads in RNA-seq. All introns in the selected 6,222 isoforms were considered. n = 2 biologically independent experiments. SS, splice sites. d, The change in expression of transcripts of five genes in WT or CDK11(G579S) HCT116 cells that were either treated with DMSO or with 50 nM OTS964 for 4 h. mRNA levels were normalized to PPIA mRNA and expression in DMSO was set as 1. n = 4 biologically independent experiments. Data are mean ± s.e.m. E–E and E–In indicate primers spanning exon–exon and exon–intron junctions, respectively. e, Schematic of the 4SU–seq experiment. f, The ratio of intronic over exonic read counts per kilobase of length (RPK) in the 4SU–seq pulse (left) or chase (right) experiment. Two biological replicates (Rep. 1 and Rep. 2) are shown. g, Comparison of the intron ratios in individual genes between DMSO-treated and either OTS964-treated (left) or pladi B-treated (right) cells in the 4SU–seq chase experiment. h, Comparison of the log2-transformed fold changes in intron ratios for individual genes in pladi B/DMSO and OTS964/DMSO in the 4SU–seq chase experiment. Differentially affected genes are indicated by circles. For the box plots in c and f, the box limits represent the range between the first and third quartiles for each condition, the centre lines show the median, and the ends of the whiskers extend to 1.5× the interquartile range.

Article Snippet: The Dynabeads were then incubated in 1 ml of buffer A with 3 μg of SF3B1 (MBL MB-D221-3), 3 μg of CDK11 (Abcam, ab19393) or 3 μg of IgG control antibodies (Proteintech, 66360- 2-Ig, 30000-0-AP) at 4 °C for 3 h. Clarified cell extracts (10,000g for 10 min) were then rotated with antibody-coated Dynabeads for 2 h at 4 °C, and were subsequently washed three times with 1 ml of buffer A. Proteins were eluted by addition of 55 μl of 3× Laemmli buffer and being boiled for 5 min. After SDS–PAGE, the following antibodies were used for detection: SF3B1 (MBL, MB-D221-3, 1:2,000), CDK11 (rabbit antiserum, 1:3,000), SF3B4 (Bethyl, A303-950A, 1:2,000), CDK12 (Santa Cruz, sc-81834, 1:1,000), CDK9 (Santa Cruz, sc-484, 1:1,000), CDK1 (Cell Signaling, 77055S, 1:500), CDK2 (Santa Cruz, sc-163, 1:1,000) and CK2a (BD Biosciences, 611610, 1:2,000).

Techniques: Activity Assay, RNA Sequencing, Expressing, Comparison, Transformation Assay

Journal: Nature

Article Title: CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

doi: 10.1038/s41586-022-05204-z

Figure Lengend Snippet: Fig. 1 | OTS964 is a highly selective CDK11 inhibitor. a, Kinome tree representation of the selectivity of OTS964 (at 1 µM concentration) in the Eurofins panel of 412 human kinases. The size of the red circles shows the percentage of inhibition of kinase activity. The green circle corresponds to CDK11. The percentage of CDK11 inhibition was derived from IVKAs presented in b and Fig. 3f. The blue circle corresponds to TOPK. The percentage of TOPK inhibition was estimated from the published IC50 = 353 nM (ref. 24) and Extended Data Fig. 1b. b, Immunoblots of IVKAs of Flag-tagged (F) CDK11 WT, G579S and kinase dead (KD) mutants (left), or Flag-tagged CDK9 WT and KD mutant (right) phosphorylation of glutathione-S-transferase (GST)-tagged RNAPIICTD substrate with the indicated concentrations of OTS964. c, The percentage of normalized NanoBRET ratio for CDK11/cyclin L2 or CDK9/cyclin T1 after OTS964 or control dinaciclib treatment. n = 2 biologically independent replicates; a representative replicate is shown. d, Immunoblot analysis of proteins after treatment of cells with OTS964 for 4 h. Short and long indicate short and long exposures of the film.

Article Snippet: The Dynabeads were then incubated in 1 ml of buffer A with 3 μg of SF3B1 (MBL MB-D221-3), 3 μg of CDK11 (Abcam, ab19393) or 3 μg of IgG control antibodies (Proteintech, 66360- 2-Ig, 30000-0-AP) at 4 °C for 3 h. Clarified cell extracts (10,000g for 10 min) were then rotated with antibody-coated Dynabeads for 2 h at 4 °C, and were subsequently washed three times with 1 ml of buffer A. Proteins were eluted by addition of 55 μl of 3× Laemmli buffer and being boiled for 5 min. After SDS–PAGE, the following antibodies were used for detection: SF3B1 (MBL, MB-D221-3, 1:2,000), CDK11 (rabbit antiserum, 1:3,000), SF3B4 (Bethyl, A303-950A, 1:2,000), CDK12 (Santa Cruz, sc-81834, 1:1,000), CDK9 (Santa Cruz, sc-484, 1:1,000), CDK1 (Cell Signaling, 77055S, 1:500), CDK2 (Santa Cruz, sc-163, 1:1,000) and CK2a (BD Biosciences, 611610, 1:2,000).

Techniques: Concentration Assay, Inhibition, Activity Assay, Derivative Assay, Western Blot, Mutagenesis, Phospho-proteomics, Control

Journal: Nature

Article Title: CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

doi: 10.1038/s41586-022-05204-z

Figure Lengend Snippet: Fig. 3 | CDK11 phosphorylates SF3B1 on threonine residues required for spliceosome activation. a, Denaturing gel analyses of the radiolabelled AdML pre-mRNA and spliced products from in vitro splicing reactions in HeLa nuclear extracts after the indicated treatments. Schematics of the intron lariat, unspliced substrate, spliced product and intron (from top to bottom) are shown on the left. −ATP, ATP-depleted nuclear extracts; M, marker; nt, nucleotides. b, Native gel analyses of the kinetics of spliceosome assembly on radiolabelled AdML pre-mRNA in HeLa nuclear extracts after the indicated treatments (top). The identities of spliceosome complexes E, A, B and C are shown. Bottom, immunoblot of proteins after the indicated treatments of the Hela nuclear extracts. c, Silver-stained SDS–PAGE gel showing immunoprecipitates of Flag-tagged empty vector (F–EV) and Flag-tagged CDK11 with or without RNase A treatment. The identity of specific bands is marked. d, Immunoblot analyses of immunoprecipitations of endogenous SF3B1. IgG, antibody control. e, Immunoblot analysis of proteins after treatment of WT or CDK11(G579S) HCT116 cells with OTS964. f, Immunoblots of IVKAs with Flag-tagged CDK11 WT, G579S and KD mutants on GST– SF3B1(1–463) substrate with the indicated concentrations of OTS964.

Article Snippet: The Dynabeads were then incubated in 1 ml of buffer A with 3 μg of SF3B1 (MBL MB-D221-3), 3 μg of CDK11 (Abcam, ab19393) or 3 μg of IgG control antibodies (Proteintech, 66360- 2-Ig, 30000-0-AP) at 4 °C for 3 h. Clarified cell extracts (10,000g for 10 min) were then rotated with antibody-coated Dynabeads for 2 h at 4 °C, and were subsequently washed three times with 1 ml of buffer A. Proteins were eluted by addition of 55 μl of 3× Laemmli buffer and being boiled for 5 min. After SDS–PAGE, the following antibodies were used for detection: SF3B1 (MBL, MB-D221-3, 1:2,000), CDK11 (rabbit antiserum, 1:3,000), SF3B4 (Bethyl, A303-950A, 1:2,000), CDK12 (Santa Cruz, sc-81834, 1:1,000), CDK9 (Santa Cruz, sc-484, 1:1,000), CDK1 (Cell Signaling, 77055S, 1:500), CDK2 (Santa Cruz, sc-163, 1:1,000) and CK2a (BD Biosciences, 611610, 1:2,000).

Techniques: Activation Assay, In Vitro, Marker, Western Blot, Staining, SDS Page, Plasmid Preparation, Control

Journal: Nature

Article Title: CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

doi: 10.1038/s41586-022-05204-z

Figure Lengend Snippet: Fig. 2 | CDK11 kinase activity is needed for efficient pre-mRNA splicing. a, Differentially expressed genes from nuclear RNA-seq in HCT116 cells treated with 30 nM OTS964 for 4 h. Downregulated and upregulated genes are shown in red and blue, respectively. Padj, adjusted P. b, IGV genome browser view of WEE1 from RNA-seq. c, The ratio of spliced reads over total unspliced and spliced reads in RNA-seq. All introns in the selected 6,222 isoforms were considered. n = 2 biologically independent experiments. SS, splice sites. d, The change in expression of transcripts of five genes in WT or CDK11(G579S) HCT116 cells that were either treated with DMSO or with 50 nM OTS964 for 4 h. mRNA levels were normalized to PPIA mRNA and expression in DMSO was set as 1. n = 4 biologically independent experiments. Data are mean ± s.e.m. E–E and E–In indicate primers spanning exon–exon and exon–intron junctions, respectively. e, Schematic of the 4SU–seq experiment. f, The ratio of intronic over exonic read counts per kilobase of length (RPK) in the 4SU–seq pulse (left) or chase (right) experiment. Two biological replicates (Rep. 1 and Rep. 2) are shown. g, Comparison of the intron ratios in individual genes between DMSO-treated and either OTS964-treated (left) or pladi B-treated (right) cells in the 4SU–seq chase experiment. h, Comparison of the log2-transformed fold changes in intron ratios for individual genes in pladi B/DMSO and OTS964/DMSO in the 4SU–seq chase experiment. Differentially affected genes are indicated by circles. For the box plots in c and f, the box limits represent the range between the first and third quartiles for each condition, the centre lines show the median, and the ends of the whiskers extend to 1.5× the interquartile range.

Article Snippet: Kinase reactions were incubated at 30 °C for 1 h. The reactions were stopped by adding 15 μl of 3× Laemmli buffer and boiled for 3 min. Kinase reactions were run on SDS–PAGE and the following antibodies were used for detection: Flag (Sigma-Aldrich, F3165, 1:3,000), phosphorylated Ser2 (Chromotek, 3E10, 1:500), phosphorylated Ser5 (Chromotek, 3E8, 1:500), GST (Santa Cruz, sc-138, 1:200), CDK9 (Santa Cruz, sc-484, 1:1,000), cyclin T1 (Santa Cruz, sc-10750, 1:2,000), CDK11 (rabbit antiserum, 1:3,000), SF3B1 phosphorylated Thr313 (Cell Signaling, 25009S, 1:2,000) and SF3B1 phosphorylated Thr235 (rabbit antiserum, 1:1,000).

Techniques: Activity Assay, RNA Sequencing, Expressing, Comparison, Transformation Assay

Journal: Nature

Article Title: CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

doi: 10.1038/s41586-022-05204-z

Figure Lengend Snippet: Fig. 1 | OTS964 is a highly selective CDK11 inhibitor. a, Kinome tree representation of the selectivity of OTS964 (at 1 µM concentration) in the Eurofins panel of 412 human kinases. The size of the red circles shows the percentage of inhibition of kinase activity. The green circle corresponds to CDK11. The percentage of CDK11 inhibition was derived from IVKAs presented in b and Fig. 3f. The blue circle corresponds to TOPK. The percentage of TOPK inhibition was estimated from the published IC50 = 353 nM (ref. 24) and Extended Data Fig. 1b. b, Immunoblots of IVKAs of Flag-tagged (F) CDK11 WT, G579S and kinase dead (KD) mutants (left), or Flag-tagged CDK9 WT and KD mutant (right) phosphorylation of glutathione-S-transferase (GST)-tagged RNAPIICTD substrate with the indicated concentrations of OTS964. c, The percentage of normalized NanoBRET ratio for CDK11/cyclin L2 or CDK9/cyclin T1 after OTS964 or control dinaciclib treatment. n = 2 biologically independent replicates; a representative replicate is shown. d, Immunoblot analysis of proteins after treatment of cells with OTS964 for 4 h. Short and long indicate short and long exposures of the film.

Article Snippet: Kinase reactions were incubated at 30 °C for 1 h. The reactions were stopped by adding 15 μl of 3× Laemmli buffer and boiled for 3 min. Kinase reactions were run on SDS–PAGE and the following antibodies were used for detection: Flag (Sigma-Aldrich, F3165, 1:3,000), phosphorylated Ser2 (Chromotek, 3E10, 1:500), phosphorylated Ser5 (Chromotek, 3E8, 1:500), GST (Santa Cruz, sc-138, 1:200), CDK9 (Santa Cruz, sc-484, 1:1,000), cyclin T1 (Santa Cruz, sc-10750, 1:2,000), CDK11 (rabbit antiserum, 1:3,000), SF3B1 phosphorylated Thr313 (Cell Signaling, 25009S, 1:2,000) and SF3B1 phosphorylated Thr235 (rabbit antiserum, 1:1,000).

Techniques: Concentration Assay, Inhibition, Activity Assay, Derivative Assay, Western Blot, Mutagenesis, Phospho-proteomics, Control

Journal: Nature

Article Title: CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1.

doi: 10.1038/s41586-022-05204-z

Figure Lengend Snippet: Fig. 3 | CDK11 phosphorylates SF3B1 on threonine residues required for spliceosome activation. a, Denaturing gel analyses of the radiolabelled AdML pre-mRNA and spliced products from in vitro splicing reactions in HeLa nuclear extracts after the indicated treatments. Schematics of the intron lariat, unspliced substrate, spliced product and intron (from top to bottom) are shown on the left. −ATP, ATP-depleted nuclear extracts; M, marker; nt, nucleotides. b, Native gel analyses of the kinetics of spliceosome assembly on radiolabelled AdML pre-mRNA in HeLa nuclear extracts after the indicated treatments (top). The identities of spliceosome complexes E, A, B and C are shown. Bottom, immunoblot of proteins after the indicated treatments of the Hela nuclear extracts. c, Silver-stained SDS–PAGE gel showing immunoprecipitates of Flag-tagged empty vector (F–EV) and Flag-tagged CDK11 with or without RNase A treatment. The identity of specific bands is marked. d, Immunoblot analyses of immunoprecipitations of endogenous SF3B1. IgG, antibody control. e, Immunoblot analysis of proteins after treatment of WT or CDK11(G579S) HCT116 cells with OTS964. f, Immunoblots of IVKAs with Flag-tagged CDK11 WT, G579S and KD mutants on GST– SF3B1(1–463) substrate with the indicated concentrations of OTS964.

Article Snippet: Kinase reactions were incubated at 30 °C for 1 h. The reactions were stopped by adding 15 μl of 3× Laemmli buffer and boiled for 3 min. Kinase reactions were run on SDS–PAGE and the following antibodies were used for detection: Flag (Sigma-Aldrich, F3165, 1:3,000), phosphorylated Ser2 (Chromotek, 3E10, 1:500), phosphorylated Ser5 (Chromotek, 3E8, 1:500), GST (Santa Cruz, sc-138, 1:200), CDK9 (Santa Cruz, sc-484, 1:1,000), cyclin T1 (Santa Cruz, sc-10750, 1:2,000), CDK11 (rabbit antiserum, 1:3,000), SF3B1 phosphorylated Thr313 (Cell Signaling, 25009S, 1:2,000) and SF3B1 phosphorylated Thr235 (rabbit antiserum, 1:1,000).

Techniques: Activation Assay, In Vitro, Marker, Western Blot, Staining, SDS Page, Plasmid Preparation, Control