EMBO/SourceData huggingface dataset:
Figure 2B. In vitro exoribonuclease assay using 5′ radiolabeled RNA substrate and immunopurified wild-type (WT) or catalytic mutant (CM) FLAG-dmDis3l2 (mutation indicated in Figure 1C), incubated for the indicated time and separated on a 15 % polyacrylamide gel followed by phosphorimaging. Quantification of degraded substrate in percent is indicated.C. Change in abundance of 256 different substrate RNAs as determined by high-throughput sequencing of substrates in experiment shown in (B).D. Decay rates of 256 different substrate RNAs. Data shown in (C) was normalized to overall decrease in substrate abundance as determined by phosphorimaging (as shown in B) and fit to the indicated model for exponential decay. Apparent decay rate of all substrates, as determined by phosphorimaging is indicated in red.E. Selected examples for individual substrates shown in (D). Values report the observed decay rate (kobs) in min-1.F. Overview of decay rates (kobs) of all 256 different substrate RNAs, as determined in (D). Error represents SEM of curve fit.G. Impact of RNA secondary structures, as determined by RNAfold (Gruber et al, 2008), and reported as effective free energy (EFE, kcal/mol), on decay rates (kobs) are shown. Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value was determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured substrates used for subsequent analysis are indicated (EFE groups 4 to 6).H. Nucleotide content of single-stranded RNA substrates, ranked from slow (top) to fast (bottom) decay by dmDis3l2.I. Cumulative distribution of decay rates for RNA substrates with the indicated nucleotide at each one of four randomized position at the 3′ end of the substrate RNA. P-Value was determined by Mann-Whitney test comparing individual nts against all.J. Functional coupling between two base positions within the randomized 3′ end of substrate RNA. Red squares show average increase in decay rate compared to all substrates. Black squares show no or negative effect.K. Boxplot decay rates of substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median decay rates SEM of fit are indicated.L. Decay rate of all substrates, or substrates containing no, one, two, or three uridine(s) within the randomized 3′ end of the substrate RNA. Median and quartile ranges are indicated. P-values were determined by Mann-Whitney test. Substrate containing 4 uridines (4U) is indicated.
Figure 3A. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003, immunopurified dmDis3l2 and wild-type (WT) or catalytic mutant (CM) Tailor. Reactions were incubated for the indicated time and separated by 15% denaturing polyacrylamide gel electrophoresis and subjected to phosphorimaging.B. Quantification of at least three independent replicates of experiment shown in A. Data represent mean SD. Half-life was determined by fitting data to single exponential (dmDis3l2 and dmDis3l2/TailorCM) or sigmoidal (dmDis3l2/TailorWT) reaction kinetics.C. RNA decay assay using synthetic, 5′ radiolabeled pre-miR-1003 containing the indicated number or uridine residues at the 3′ end and immunopurified dmDis3l2.D. Quantification of three independent replicates of experiment shown in C. Data represent mean SD. Data were fit to single exponential reaction kinetics.E. Reaction kinetics (kobs) determined in D plotted against number of 3′ terminal uridine residues. Error of fit is shown as SEM.
Figure 4A and E. Western blot analysis of genetically modified Drosophila S2 cells. Unmodified S2 cells (wild-type, wt), or S2 cells depleted of Tailor (tailorko) by CRISPR/Cas9 or expressing FLAG-tagged Tailor (TailorOE) were analyzed. Antibodies are indicated. Note, that panels were assembled from two technical replicates of identical lysates (see source data).B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.D and H. Quantification of at least five (D) or seven (H) independent replicates of experiment shown in B/C and F/G, respectively. Uridylation signal intensity for substrate S1 (indicated in B and F) was corrected for adenylation activity and normalized to wild-type 2 min time point. Data represent mean SEM. P-values were determined using Student's t-test. * p<0.05, ** p<10-2, *** p<10-3, **** p<10-4.A and E. Western blot analysis of genetically modified Drosophila S2 cells. Unmodified S2 cells (wild-type, wt), or S2 cells depleted of dmDis3l2 (dmDis3l2ko) by CRISPR/Cas9 or expressing FLAG-tagged catalytic mutant dmDis3l2 (dmDis3l2CM OE) were analyzed. Antibodies are indicated. Note, that panels were assembled from two technical replicates of identical lysates (see source data).B, C, F and G. Lysate of S2 cells (as characterized in A and E) were incubated with -32P-UTP (B and F) or -32P-ATP (C and G) for the indicated time followed by RNA extraction, denaturing polyacrylamide gel electrophoresis and phosphorimaging.D and H. Quantification of at least five (D) or seven (H) independent replicates of experiment shown in B/C and F/G, respectively. Uridylation signal intensity for substrate S1 (indicated in B and F) was corrected for adenylation activity and normalized to wild-type 2 min time point. Data represent mean SEM. P-values were determined using Student's t-test. * p<0.05, ** p<10-2, *** p<10-3, **** p<10-4.
Figure 5A. Protein/RNA co-immunopurification experiments. FLAG-tagged versions of indicated proteins were expressed in S2 cells, followed by immunopurification and RNA isolation. RNA was visualized by CIP/PNK treatment using -32P-ATP followed by denaturing polyacrylamide gel electrophoresis and phosphorimaging. Star indicates non-specific contaminant.
Figure 6B. Meta-analysis of 3′ end counts in dmDis3l2CM-IP mapping to the indicated region around the annotated 3′ end of 68 tRNA loci in the Drosophila melanogaster genome. Cumulative relative distribution of 3′ end signal, relative to the annotated 3′ end, is reported (bottom). The average genome thymine (T)-content for the same regions is displayed (top).C. Northern hybridization experiment using total RNA from adult whole male flies using probes against 3′ trailer or mature tRNAAla(TGC). Wild-type flies (w1118), or flies bearing a homozygous frame-shift mutation in the first coding exon (dmDis3l2-/-) or a homozygous amino acid-exchange mutation in the catalytic site (dmDis3l2CM) in the endogenous dmdis3l2 locus in the same genetic background were used. Probes against 2S rRNA served as loading control.D. Quantification of four independent biological replicates of experiment shown in C. P-Value determined by Student's t-test.E. Exoribonuclease assay using immunopurified dmDis3l2 and in vitro transcribed, 5′ radiolabeled mature tRNA, containing a CCA modification or 3′ trailer-containing tRNA. Reactions were incubated for the indicated time and subjected to polyacrylamide gel electrophoresis and phosphorimaging.F. Quantification of three independent replicates of experiment shown in E. Data represent mean SD. Decay rates (kobs) SEM of fit were determined upon fitting to single exponential decay kinetics.
Figure 7A. Impact of RNA secondary structures on RNA decay rates (kobs) of 256 different RNA substrates subjected to E.coli RNase R (ecoRNaseR)- or Drosophila melanogaster Dis3l2 (dmDis3l2)-directed RNA decay assay as described in Fig. 2. RNA secondary structures were determined by RNAfold (Gruber et al, 2008) and reported as effective free energy (EFE). Error bars represent SEM of curve fit. Grey boxes represent tukey boxplot of individual EFE groups. White line indicates median. P-Value determined by Mann-Whitney test comparing individual EFE groups against all substrates. Unstructured RNA used for subsequent analysis are indicated (EFE groups 4 to 6).B. Nucleotide content of randomized 3′ end sequence among the indicated number of substrate RNAs, ranked according to decay kinetics from slow (left) to fast (right). Fold-change in abundance for individual nucleotides is reported (bottom). Only unstructured RNA substrates (see A) were considered.C. Decay rate of the indicated number of sequences, grouped according to individual nucleotide content in the randomized 3′ end sequence. Data is represented as boxplot, outliers are not shown. P-Value reports significant differences compared to all substrates as determined by Mann-Whitney test. Grey area represents the inner quartile range and white line the median of all substrates.
Figure 1(C) HeLa GFP-LC3 cells were transfected with indicated siRNAs and infected with DsRed-labeled Salmonella. The percentage of GFP-LC3+ Salmonella at 1 hr post-infection is shown. Data are shown as means ± SD; n = 75 cells per condition from three biological replicates. Data are representative of three independent experiments.(D) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in (C). The scale bars represent 10 μm.(E) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments.(G) HeLa GFP-LC3 cells were transfected with control siRNA, CLEC12A siRNA1, or CLEC12A siRNA2 as well as CLEC12A rescue constructs (R1 or R2) and infected for 1 hr with DsRed-labeled Salmonella. Data are shown as mean ± SD; n = 75 per condition. Data are representative of three independent experiments.(H) HeLa cells from (G) were lysed, and HA-CLEC12A expression levels were assessed by western blot. Data are representative of three independent experiments.
Figure 2(A) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA. The scale bars represent 10 μm. (B) Percentage of LC3-Salmonella colocalization in WT HeLa cells, ATG16L1 KO HeLa cells, and ATG16L1 KO HeLa cells stably expressing ATG16L1∗300T or ∗300A alleles in the presence of control siRNA or CLEC12A siRNA. Data are shown as means ± SD; n = 75. Data shown are representative of three independent experiments.
Figure 3(A) Confocal micrographs of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs at 1 hr post-infection. The scale bars represent 10 μm. (B) Quantification of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs. Data shown represent mean ± SD of n = 3 independent experiments; unpaired t test. ∗∗p < 0.01.(C and D) Salmonella cfus quantified per gram of stool (C) and per organ (D) at 4 days post-infection are displayed. Data are shown as mean ± SD; ∗∗p ≤ 0.01. Data are representative of at least two independent experiments (n = 9 for WT; n = 7 or 8 for Clec12a−/−).(E) Clinical score for infected mice at 4 days post-infection. Data are shown as mean ± SD. Data are representative of at least two independent experiments. ∗∗∗∗p < 0.0001; unpaired t test. (n = 14 for WT; n = 13 for Clec12a−/−).(F) Survival curve for infected WT and Clec12a−/− mice (n = 9 mice per genotype).
Figure 4(A) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs. Data are shown as mean ± SD; n = 6. Data are representative of four independent experiments.(B) Quantitation of CLEC12A-bacteria and LC3-bacteria colocalization at indicated time points. Data are shown as mean ± SD; n = 3 independent experiments. (C) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP-CLEC12A at 20, 40, 60, and 80 min post-infection. The scale bars represent 10 μm.
Figure 5(A-C) Representative epifluorescent images of HeLa cells showing colocalization of (A) CLEC12A (green) and galectin 8 or galectin 3 (red) around intracellular Salmonella (blue), (B) GFP-CLEC12A (green) and NDP52 (red) around Salmonella (blue), and (C) endogenous CLEC12A (red) and GFP-LC3 (green) around Salmonella (blue).(D) CLEC12A or GFP-CLEC12A (green) and GABARAPL2 or ATG16L1 (red) around intracellular Salmonella (blue). Images are representative of three or more independent experiments.(E) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed-labeled Salmonella for 1 hr. (F) Quantitation of ubiquitin-bacteria colocalization at 1 hr post-infection in cells shown in (E). Data are shown as means ± SEM from three independent experiments.(G) Representative epifluorescent images of HeLa cells transfected and infected as in (E). (H) Quantitation of NDP52-bacteria colocalization at 1 hr post-infection in cells shown in (G). Data are shown as means ± SEM from three independent experiments.(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.
Figure 6(E) HeLa GFP-LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed-labeled Salmonella for 1 hr. The fraction of GFP-positive intracellular Salmonella is shown. Data are mean ± SD and are representative of three independent experiments.(F) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1, KLHL9, NEDD8, or KLHL13. Data shown as mean ± SD; n = 4. Data are representative of three independent experiments.
Figure 1B Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS (top right) and OS (bottom right) of breast cancer patients in the METABRIC dataset. Genes located at the HER2 amplicon were nominated according to hazard-ratio levels (all P-values < 0.01). The HER2 co-amplification percentage of the indicated genes is represented as bar graphs (left).C The frequency of CDK12 amplification in HER2-amplified- and HER2-nonamplified breast cancer. The bar graphs indicate the frequency of HER2 and CDK12 co-amplified cases among the patients with HER2 gene amplification (left). Tables show the percentage of CDK12 amplification in the indicated cohorts (right). Amp, amplification; non-amp, non-amplification.D Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as the Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). P-values were calculated using one-way ANOVA, followed by post-hoc LSD test.E Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan-Meier method with the log-rank test.
Figure 3A Heatmap presents the alteration of gene expression (≥1.5-fold) between control (CON) and CDK12-overexpressing (CDK12) ZR-75-30 cells obtained from RNA-seq analysis.B GSEA displaying correlations of the gene-expression profiles between control and CDK12-overexpressing cells based on RNA-seq results.D The CDK12-target genes identified by RNA-seq were validated in CDK12-overexpressing ZR-75-30 cells using qRT-PCR analysis. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test.E ChIP-qPCR analysis displaying the fold-enrichment of the indicated proteins and histone H3 acetylation at the TSS of CDK12-target genes in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.F Effect of CDK12 kinase inhibition on the expression of WNT1, WNT3, and IRS1. Cells were treated with different doses of dinaciclib for 12 h, and the expression of candidate targets of CDK12 were analysed by immunoblot and qRT-PCR Mean ± s.d. (n = 3). P-values by ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells ; R, trastuzumab-resistant; S, trastuzumab-sensitive.G Cell viability was measured by SRB assay in trastuzumab-sensitive BT474 cells and trastuzumab-resistant HCC-1954 cells treated with 1 µg/mL or 100 µg/mL trastuzumab (Trz) and/or 5 nM or 10 nM dinaciclib (Dina), respectively. Mean ± s.d. (n = 3). P-values, RM ANOVA with a post-hoc LSD test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.H Tumor growth curves for mice xenografted with the indicated HER2+ breast cancer cells treated with 20 mg/kg trastuzumab and/or 20 mg/kg dinaciclib (n = 8/group; mean ± s.e.m.). P-values, one-way ANOVA with a post-hoc LSD (BT474) or Dunnett T3 (HCC-1954) test for the last day of tumor measurement. Cell line information: a/a, CDK12-amplified/HER2-amplified cells R, trastuzumab-resistant; S, trastuzumab-sensitive.
Figure 4A Western blot and qRT-PCR results showing phosphorylation levels of GSK-3β and WNT1 and WNT3 expression, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values by two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitiveSubcellular localization of β-catenin in the indicated stable cell lines were analysed by cellular fractionation (B)Subcellular localization of β-catenin in the indicated stable cell lines were analysed by immunofluorescence staining (C). Nuclear translocation of β-catenin in control, CDK12-overexpressing, and CDK12-knockdown stable cell lines was quantified by counting the cells harboring exclusively nuclear β-catenin (N) from the immunofluorescence image. Mean ± s.d. (n = 3). P-value was calculated using a two-tailed Student's t-test. M+C, cells containing membrane and/or cytoplasmic β-catenin; N, cells dominantly expressing nuclear β-catenin. Green, β-catenin staining; Blue, DAPI staining for visualization of nucleus; Scale bar = 10 μ. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive.D Luciferase-reporter assay measuring the TCF/ lymphoid enhancer-binding factor-promoter activity in the indicated cell lines. TOP, pTOP-flash (wild-type TCF-binding sites), FOP, pFOP-flash (mutant TCF-binding sites). Data represent the mean ± s.d. P-values were calculated using a two-tailed Student's t-test.E The mRNA levels of the WNT/β-catenin/TCF-target genes in CDK12-overexpressing ZR-75-30 cells and CDK12-knockdown BT474 cells were analysed by qRT-PCR. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test.F Effect of WNT/ β-catenin/TCF pathway on the CDK12 regulation of breast CSCs. Cells were treated with DMSO (vehicle) or 10 nM FH535, a β-catenin/TCF inhibitor, for 24 h, and changes in the CD44+/CD24-/ESA+ cell population were measured using FACS analysis. Cells seen at lower right quadrant in the dot plots indicate CD44+/CD24- population (red) and CD44+/CD24-/ESA+ population (blue). Data represent the mean ± s.d. ofP-value was calculated using one-way ANOVA with a post-hoc LSD test.
Figure 5A Human phospho-RTK array displaying the activation of phosphokinase-related proteins in CDK12-knockdown BT474 cells. The bar graph shows the fold change of spot intensity. Cell line information: a/a, CDK12-amplified/HER2-amplified cells S, trastuzumab-sensitive.B Immunoblots showing the levels of the indicated proteins in different types of HER2+ breast cancer stably expressing CDK12 or with CDK12 knocked down. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive.Co-IP analysis showing the amounts of bindings between ErbB receptors. Cell lysates from CDK12-overexpressing ZR-75-30 cells were immunoprecipitated with anti-HER2 antibody and immunoblotted using the indicated antibodies. IgG, negative control. Cell line information: ; n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.D Western blot and qRT-PCR analysis of the phosphorylation levels and expression of IRS1, respectively, in the indicated cell lines. Mean ± s.d. (n = 3). P-values were calculated using a two-tailed Student's t-test. Cell line information: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant S, trastuzumab-sensitive.E Co-IP analysis indicating the amounts of IRS1 protein interaction with ErbB receptors and p85 in CDK12-overexpressing ZR-75-30 cells. Cell line information n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.F IRS1 effect on CDK12-induced phosphorylation of ErbB receptors. Lysates from cells treated with either control siRNA or IRS1 siRNA (siIRS1) for 24 h were subjected to immunoblotting with the indicated antibodies. Cell line information: n/a, CDK12 non-amplified/HER2-amplified cells S, trastuzumab-sensitive.
Figure 1(B-F) Basal views through living SCs, with dashed white circles approximating the outline of a single SC, and acidic compartments marked by the vital dye LysoTracker® Red (magenta). In merge images, a single non-acidic (C, D, F) and acidic (E) compartment containing intraluminal vesicles (ILVs) is boxed and magnified in the right panel (Zoom). ILVs appear as membrane-delineated vesicles, using super-resolution 3D-structured illumination (3D-SIM) microscopy for the brighter overexpressed GFP-tagged constructs (yellow; C and F). However, ILVs appear only as puncta, using lower resolution wide-field microscopy for the fainter endogenously expressed YFP-tagged Rab GTPases (yellow; D and E). (B) Wide-field fluorescence image, including differential interference contrast (DIC), of SC expressing a GFP-tagged version of human CD63 (CD63-GFP). CD63-GFP expression is apparent on the limiting membranes of non-acidic compartments and their ILVs, and also on the limiting membranes of the enlarged acidic compartments. Most large non-acidic compartments are Rab11-positive (D) and contain dense-core granules, which have a 'fried egg' appearance with DIC (arrowheads) (Corrigan et al., 2014; Redhai et al., 2016). (C) 3D-SIM image of CD63-GFP-expressing SC. Arrow highlights CD63-GFP-marked ILVs (Zoom). Many more ILVs are apparent in non-acidic compartments in a complete Z-stack of a non-acidic compartment (Movie EV1). (D) Wide-field fluorescence image of an SC expressing a YFP-Rab11 gene trap. YFP-Rab11 marks the limiting membranes of most non-acidic compartments and internal puncta (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1B). (E) Wide-field fluorescence image of SC expressing a YFP-Rab7 gene trap. YFP-Rab7 marks the limiting membranes of acidic compartments and internal puncta (arrow in Zoom). Enlarged acidic compartments are also present in adjacent main cells. (F) 3D-SIM image of SC expressing a GFP-tagged version of Breathless (Btl-GFP). Btl-GFP marks the limiting membranes of non-acidic compartments and their ILVs (arrow in Zoom), but not the surface of acidic compartments (Appendix Fig S1C). Images from six-day-old male flies shifted to 29°C at eclosion. This induces GAL4/UAS-dependent SC transgene expression in (B), (C) and (F). The genotypes of flies carrying multiple transgenes are: w; P[w+, UAS-CD63-GFP] P[w+, tub-GAL80ts]/+; dsx-GAL4/+ (B, C); w; P[w+, tub-GAL80ts]/+; dsx-GAL4/P[w+, UAS-btl-GFP] (F).