Journal: Nucleic Acids Research

Article Title: DDX24 modulates angiogenesis by promoting CCR4-NOT complex-dependent mRNA decay

doi: 10.1093/nar/gkag130

Figure Lengend Snippet: DDX24 regulates endothelial functions through its RNA-binding activity. ( A ) Schematic illustration of the domain architecture of DDX24. WT, wild type; RBD, RNA-binding domain. DDX24-ΔRBD (amino acids 395–448 deleted) is shown at the bottom. ( B, C ) qPCR quantification ( B ) and immunoblot analysis ( C ) of FLAG-tagged DDX24-WT or DDX24-ΔRBD overexpression in HUVECs. ( D ) Measurement of rRNA enrichment in HUVECs by RIP-qPCR. HUVECs were transfected with FLAG-tagged DDX24-WT or DDX24-ΔRBD, RNA–protein complexes were immunoprecipitated with FLAG or IgG (negative control). Results are presented relative to input. ( E ) Binding affinity of DDX24-WT or DDX24-ΔRBD to 5.8S rRNA measured by BLI. DDX24-WT or DDX24-ΔRBD binding to immobilized 5.8S rRNA was tested using a range of protein concentrations from 125 to 1000 nM in two-fold dilution. The affinity constants (KD) determined by kinetic analysis are provided. ( F, G ) Transwell assay showing the rescue of DDX24 knockdown effects on HUVEC migration ( F ) and invasion ( G ) with siRNA-resistant DDX24-WT or DDX24-ΔRBD. Scale bar, 200 μm. Quantification of the migrated or invasive cells was counted and plotted. ( H ) Rescue of DDX24 knockdown effects on HUVEC tube formation on Matrigel with siRNA-resistant DDX24-WT or DDX24-ΔRBD. Scale bar, 200 μm. Quantification of branch points was counted and plotted. Data are presented as mean ± SEM and are representative of three independent experiments; the P -values were calculated using Bonferroni–Šídák multiple comparisons test. * P <.05; ** P <.01; *** P <.001; ns, not significant.

Article Snippet: The RNA was reverse-transcribed using HiScript ® II Q RT SuperMix (Vazyme) and following quantitative polymerase chain reaction (qPCR) analysis was performed using TB Green premix ExTaq Real-Time polymerase chain reaction (PCR) kit (Takara) on an ABI QuantStudio 7 Flex PCR system (Applied Biosystems) with primers listed in .

Techniques: RNA Binding Assay, Activity Assay, Western Blot, Over Expression, Transfection, Immunoprecipitation, Negative Control, Binding Assay, Transwell Assay, Knockdown, Migration

Journal: Nucleic Acids Research

Article Title: DDX24 modulates angiogenesis by promoting CCR4-NOT complex-dependent mRNA decay

doi: 10.1093/nar/gkag130

Figure Lengend Snippet: DDX24 directly binds to angiogenesis-associated mRNAs in endothelial cells. ( A ) Workflow for DDX24 irCLIP-seq. ( B ) Distribution of DDX24 CLIP-seq peaks over known genomic features. CDS, coding sequence; UTR, untranslated regions; ncRNA, noncoding RNA. ( C ) Top 10 enriched Gene Ontology (GO) biological processes associated with the irCLIP-seq identified DDX24-bound mRNAs. ( D ) Measurement of CLEC14A, ERG and RPL12 (nontarget control) mRNA enrichment in HUVECs by RIP-qPCR. HUVECs were transfected with FLAG-tagged DDX24-WT or DDX24-ΔRBD, and RNA–protein complexes were immunoprecipitated with FLAG or IgG (negative control). Results are presented relative to input, as mean ± SEM ( n = 3); the P -values were calculated using Bonferroni–Šídák multiple comparisons test. ** P <.01; *** P <.001; ns, not significant. ( E ) RNA pulldown assay using tRSA and tRSA-fused CLEC14A or ERG RNAs incubated with HEK293T lysates transfected with FLAG-tagged DDX24-WT or DDX24-ΔRBD, followed by immunoblot analysis with the indicated antibodies. ( F ) Binding affinity of DDX24-WT and DDX24-ΔRBD to CLEC14A RNA (left) or ERG RNA (right) measured by BLI. DDX24-WT or DDX24-ΔRBD binding to immobilized RNA was tested using a range of protein concentrations from 125 to 1000 nM (in two-fold dilution). The affinity constants (KD) determined by kinetic analysis are provided.

Article Snippet: The RNA was reverse-transcribed using HiScript ® II Q RT SuperMix (Vazyme) and following quantitative polymerase chain reaction (qPCR) analysis was performed using TB Green premix ExTaq Real-Time polymerase chain reaction (PCR) kit (Takara) on an ABI QuantStudio 7 Flex PCR system (Applied Biosystems) with primers listed in .

Techniques: Sequencing, Control, Transfection, Immunoprecipitation, Negative Control, Incubation, Western Blot, Binding Assay

Journal: Nucleic Acids Research

Article Title: DDX24 modulates angiogenesis by promoting CCR4-NOT complex-dependent mRNA decay

doi: 10.1093/nar/gkag130

Figure Lengend Snippet: DDX24 mediates endothelial cell function by downregulating key angiogenesis-related genes. ( A ) Cumulative fraction of mRNA level changes between siDDX24 and siNC for nontargets and DDX24 targets. P -values were calculated using two-sided Wilcoxon and Mann–Whitney test. ( B ) Binding percentage of the RNA-seq DEGs in cells upon DDX24 knockdown. DDX24 bound or unbound mRNAs are identified by irCLIP-seq. ( C ) Top 10 enriched GO biological processes with the mRNAs that upregulated in RNA-seq upon DDX24 knockdown as well as DDX24 bound identified in irCLIP-seq. ( D ) Volcano plot showing changes in expression levels of DDX24-bound mRNA identified by irCLIP in HUVECs transfected with siDDX24. The two transcripts of interest are indicated. ( E, F ) qPCR quantification ( E ) and immunoblot ( F ) measurements of CLEC14A and ERG in HUVECs transfected with siRNA-resistant DDX24-WT (reWT) or DDX24-ΔRBD (reΔRBD) upon DDX24 knockdown. CTL, HUVECs transfected with nonsense siRNA; KD, HUVECs transfected with DDX24 siRNA. ( G, H ) Transwell assay showing the effect of CLEC14A or ERG knockdown on the migration ( G ) and invasion ( H ) of DDX24-knockdown HUVECs. Scale bar, 200 μm. Quantification of the migrated or invasive cells was counted and plotted. ( I ) The effect of CLEC14A or ERG knockdown on the tube formation on Matrigel of DDX24-knockdown HUVECs. Scale bar, 200 μm. Quantification of branch points was counted and plotted. Data are presented as mean ± SEM and are representative of three independent experiments; the P -values were calculated by Bonferroni–Šídák multiple comparisons test. * P <.05; ** P <.01; *** P <.001; ns, not significant.

Article Snippet: The RNA was reverse-transcribed using HiScript ® II Q RT SuperMix (Vazyme) and following quantitative polymerase chain reaction (qPCR) analysis was performed using TB Green premix ExTaq Real-Time polymerase chain reaction (PCR) kit (Takara) on an ABI QuantStudio 7 Flex PCR system (Applied Biosystems) with primers listed in .

Techniques: Cell Function Assay, MANN-WHITNEY, Binding Assay, RNA Sequencing, Knockdown, Expressing, Transfection, Western Blot, Transwell Assay, Migration

Journal: Nucleic Acids Research

Article Title: DDX24 modulates angiogenesis by promoting CCR4-NOT complex-dependent mRNA decay

doi: 10.1093/nar/gkag130

Figure Lengend Snippet: DDX24 promotes the decay of its mRNA targets. ( A ) Workflow for RNA half-life determination. Transcriptome-wide mRNA stability measurements were performed after transcriptional silencing with 10 μg/ml ActD and RNA-seq analysis of poly-A + RNA extracted at regular time intervals. ( B ) Violin plot depicting half-lives of all mRNAs in siDDX24 versus siNC HUVECs. The dotted line shows the median mRNA half-life in siNC HUVECs. Box plots show minimum value, first quartile, median, third quartile and maximum value. P -value of the difference in distance between the two categories was determined by the Mann–Whitney-Wilcoxon test. ( C ) Scatter plot and marginal histograms showing mRNA half-lives in siDDX24 versus siNC HUVECs. ( D ) Cumulative frequency of log 2 fold changes in mRNA life-time between siDDX24 and siNC for nontargets and DDX24 targets. P -values were calculated using two-sided Wilcoxon and Mann–Whitney test. ( E ) The mRNA half-life of CLEC14A in HUVECs with DDX24 knockdown (KD) and subsequent restoration of siRNA-resistant DDX24-WT (reWT) or DDX24-ΔRBD (reΔRBD). RNA levels were quantified by RT-qPCR at 0, 4, 8 h and presented as mean ± SEM. The mRNA half-life calculated from the qPCR experiments was determined using nonlinear regression (one-phase decay) and plotted for statistical analysis on the right. P -values were calculated using Bonferroni–Šídák multiple comparisons test. ( F ) Diagrams illustrating the locations of DDX24 irCLIP-seq peaks on CLEC14A mRNAs. Below is the design of GFP-based mRNA reporter constructs and binding motif mutated constructs. Mut, mutant construct. ( G ) Enrichment of WT and binding motif mutated GFP-based RNA reporters by DDX24-RIP in HUVEC. HUVECs were transfected with FLAG-tagged DDX24 and CLEC14A (GFP-1) RNA reporters, RNA–protein complexes were immunoprecipitated with FLAG, then GFP and RPL12 (nontarget control) RNA enrichment was tested. Results are presented relative to input, as mean ± SEM ( n = 3); the P -values were calculated by two-tailed unpaired Student’s t -test. ( H ) The mRNA half-life of WT and mutated CLEC14A (GFP-1) reporter constructs were measured in HUVECs following DDX24 knockdown. ( I ) Polysome profiles of HUVECs with DDX24 knockdown as determined by sucrose density-gradient ultracentrifugation. The curves have no significant difference. ( J ) Cumulative distribution log 2 -fold changes of translation efficiency (ratio of ribosome bound fragments and mRNA input) between siDDX24 and siNC for nontargets (Not bound) and CLIP targets (DDX24 bound). P -values were calculated using a two-sided Mann–Whitney test. * P <.05; ** P <.01; *** P <.001; ns, not significant.

Article Snippet: The RNA was reverse-transcribed using HiScript ® II Q RT SuperMix (Vazyme) and following quantitative polymerase chain reaction (qPCR) analysis was performed using TB Green premix ExTaq Real-Time polymerase chain reaction (PCR) kit (Takara) on an ABI QuantStudio 7 Flex PCR system (Applied Biosystems) with primers listed in .

Techniques: RNA Sequencing, MANN-WHITNEY, Knockdown, Quantitative RT-PCR, Construct, Binding Assay, Mutagenesis, Transfection, Immunoprecipitation, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: DDX24 modulates angiogenesis by promoting CCR4-NOT complex-dependent mRNA decay

doi: 10.1093/nar/gkag130

Figure Lengend Snippet: DDX24 directly interacts with the CCR4-NOT complex. ( A ) Co-IP and western blotting showing interactions of FLAG-tagged DDX24 to HA-tagged CNOT2, CNOT3, CNOT1-C (C-terminal region of CNOT1) but not of CNOT1-M (middle region of CNOT1) in HEK293T cells. ( B ) Binding affinity of CNOT2, CNOT3, CNOT1-C, or CNOT1-M to DDX24 protein measured by BLI. DDX24 binding to immobilized CNOT2, CNOT3, CNOT1-C, or CNOT1-M protein was tested using a range of protein concentrations from 0.625 to 5 μM (in two-fold dilution). The affinity constants (KD) determined by kinetic analysis are provided. ( C ) Schematic model of how DDX24 enhances RNA decay. The direct interaction of DDX24 with CNOT2/3 and mRNAs drive the CCR4-NOT complex and target mRNA interaction and the subsequent mRNA decay. ( D ) Fluorescence immunostaining of FLAG-tagged DDX24 (anti-FLAG, Alexa 488) and CNOT2 (anti-CNOT2, Alexa 594) in HUVECs, nuclei were stained by DAPI. Scale bar, 8 μm. Co-localization between DDX24 and CNOT2 was characterized by Pearson’s coefficient, R2 = 0.7972, analyzed by Leica 880 software. ( E ) qPCR quantification of CLEC14A and ERG RNA levels in CNOT2-knockdown HUVECs upon DDX24 silencing ( n = 3). Data are presented as mean ± SEM, the P -values were calculated using Bonferroni–Šídák multiple comparisons test. ** P <.01; *** P <.001; ns, not significant.

Article Snippet: The RNA was reverse-transcribed using HiScript ® II Q RT SuperMix (Vazyme) and following quantitative polymerase chain reaction (qPCR) analysis was performed using TB Green premix ExTaq Real-Time polymerase chain reaction (PCR) kit (Takara) on an ABI QuantStudio 7 Flex PCR system (Applied Biosystems) with primers listed in .

Techniques: Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Fluorescence, Immunostaining, Staining, Software, Knockdown

Journal: Nucleic Acids Research

Article Title: DDX24 modulates angiogenesis by promoting CCR4-NOT complex-dependent mRNA decay

doi: 10.1093/nar/gkag130

Figure Lengend Snippet: DDX24 promotes mRNA decay in a CCR4-NOT-dependent manner. ( A ) The mRNA half-life of CLEC14A, ERG in CNOT2-knockdown HUVECs upon DDX24 silencing following transcription inhibition by 10 μg/ml ActD. ( B ) The half-life of GFP-based CLEC14A reporter RNA in CNOT2-knockdown HUVECs upon DDX24 silencing following transcription inhibition. RNA levels were quantified via RT-qPCR at 0, 6, 12 h. The mRNA half-life calculated from the qPCR experiments was determined using nonlinear regression (one-phase decay) and plotted for statistical analysis. ( C, D ) Transwell assay showing the effect of DDX24 knockdown on the migration ( C ) and invasion ( D ) in HUVECs upon CNOT2 knockdown. Scale bar, 200 μm. Quantification of the migrated or invasive cells was counted and plotted. ( E ) The effect of DDX24 knockdown on the tube formation on Matrigel in HUVECs upon CNOT2 knockdown. Scale bar, 200 μm. Quantification of branch points was counted and plotted. Data are presented as mean ± SEM from three independent experiments. P -values were calculated using Bonferroni–Šídák multiple comparisons test [panel ( A )] or by two-tailed unpaired Student’s t -tests [panels ( B ) –(E )]. * P <.05; *** P <.001; ns, not significant.

Article Snippet: The RNA was reverse-transcribed using HiScript ® II Q RT SuperMix (Vazyme) and following quantitative polymerase chain reaction (qPCR) analysis was performed using TB Green premix ExTaq Real-Time polymerase chain reaction (PCR) kit (Takara) on an ABI QuantStudio 7 Flex PCR system (Applied Biosystems) with primers listed in .

Techniques: Knockdown, Inhibition, Quantitative RT-PCR, Transwell Assay, Migration, Two Tailed Test