anti cdk12  (Thermo Fisher)

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: CDK12 restricts FHV replication but not FHVΔB2. ( A ) RT‒qPCR analysis of the mRNA levels in S2 cells treated with the indicated dsRNAs. β -gal dsRNA was used in the control. ( B and C ) S2 cells were pretreated with dsRNA against a control (β -gal ) or the indicated genes for 48 h and then transfected with pMT-B2-V5 ( B ) or pMT-GFP ( C ) plasmids for another 48 h. The pretreated cells were infected with FHVΔB2 and the FHV RNA1 levels, relative to the control, were determined by RT‒qPCR at 24 hpi. ( D ) S2 cells were pretreated with dsRNA against a control (β -gal ) or the indicated genes for 48 h and infected with FHV (MOI = 1), prior to viral RNA quantification with RT‒qPCR at 24 hpi. The relative mRNA or viral RNA levels were normalized to the controls ( A–D ). Error bars represent SD in triplicate experiments ( A–D ). Statistical analysis was performed for panels A–D ; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, n.s., not significant (Student’s t -test).

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Control, Transfection, Infection

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: CDK12 induced antiviral RNAi response by inhibiting the function of B2. ( A and B ) S2 cells were pretreated with three different dsRNAs targeting CDK12 or control dsRNA (β -gal ) for 48 h and transfected with pMT-B2-GFP or pMT-GFP plasmids for another 48 h. The GFP-positive cells were sorted by FACS and followed by FHV∆B2 infection. The total RNA was isolated at 24 hpi. The CDK12 mRNA levels ( A ) and FHV RNA1 levels ( B ), relative to the control in GFP group, were determined by RT‒qPCR. ( C ) S2 cells were pretreated with three different dsRNAs targeting CDK12 or control dsRNA (β -gal ) for 48 h and infected with FHV (MOI = 1), prior to viral RNA quantification with RT-qPCR at 24 hpi. ( D ) S2 cells were co-transfected with pMT-B2-GFP and pMT-GFP, pMT-B2-GFP and pMT-V5-CDK12, pMT-GFP and pMT-V5-CDK12, or pMT-GFP for 48 h and infected with FHVΔB2, prior to viral RNA quantification with RT‒qPCR at 24 hpi. ( E ) S2 cells transfected with pMT-V5-CDK12 and pMT-GFP for 48 h and infected with FHV (MOI = 1), prior to FHV RNA quantification with RT-qPCR at 24 hpi. ( F ) S2 cells were pretreated with three different dsRNAs targeting CDK12 or control dsRNA (β -gal ) for 48 h and then co-transfected with psi-CHECK2 plasmids and pMT-B2-GFP or pMT-GFP plasmids for another 48 h. The pretreated cells were treated with dsRNAs against firefly luciferase or β -gal , and both firefly and Renilla luciferase activity were measured at 48 h. The activity ratios between firefly luciferase and Renilla luciferase were shown and normalized to the control in dsRNA targeting β -gal group. Error bars represent SD in three independent experiments performed in triplicate. Statistical analysis was performed using ANOVA; ∗∗∗ P < 0.001, n.s., not significant.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Control, Transfection, Infection, Isolation, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: The critical amino acids responsible for dsRNA binding and dimerization in B2 were essential for its interaction with CDK12. ( A ) S2 cells co-transfected with pMT-V5-CDK12 plasmid and pMT-B2-GFP or pMT-GFP plasmids were subjected to Co-IP using anti-V5 (left) or anti-GFP (right) and western blot for the immunoprecipitates using the indicated antibodies. Black arrows indicate the target proteins. ( B ) S2 cells transfected with pMT-B2-V5 plasmid for 48 h and infected with FHVΔB2, prior to Co-IP using anti-V5 antibodies, and western blot for the precipitates using anti-CDK12, anti-V5, and anti-GAPDH antibodies. ( C ) Representative images from immunofluorescence staining of co-localization between B2 and CDK12 in S2 cells co-transfected with pMT-V5-CDK12 and pMT-B2-GFP plasmids. ( D ) Lysates from S2 cells co-expressing V5 tagged CDK12 and GFP-tag B2 were untreated or pretreated with RNase A, prior to Co-IP using anti-V5 antibodies. Western blot for the immunoprecipitates using the indicated antibodies. Black arrows indicate the target proteins. ( E ) Schematic representation of B2 containing three α-helices and three mutants deficient in key amino acids for dimerization, dsRNA binding, or Dcr-2 binding functions of B2, respectively. ( F ) S2 cells were co-transfected with pMT-V5-CDK12 plasmid and pMT-B2-GFP or GFP-tagged B2 mutants expressing plasmids, and cells without V5-CDK12 expression were used as a negative control, prior to Co-IP using anti-V5 antibody. The immunoprecipitates were subjected to western blotting using the indicated antibodies.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Infection, Immunofluorescence, Staining, Expressing, Negative Control

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: CDK12 is required for FHV-derived siRNA production. ( A and B ) Length distributions of total small RNAs ( A ) or FHV-derived small RNAs ( B ) sequenced from wild-type or CDK12 knockdown cells infected with FHV (MOI = 1) at 24 hpi. The yellow shade indicated small RNA peaks. ( C and E ) Distributions of each size of FHV-derived small RNA aligned to the positive and negative strands of the FHV genome in wild-type cells ( C ) or CDK12 knockdown cells ( E ). blue, positive-stranded vsiRNAs; red, negative-stranded vsiRNAs. ( D and F ) The distribution of 21 nt-long vsiRNAs reads in the positive and negative-stranded FHV RNA1 and the relative abundances of positive and negative-stranded vsiRNAs (counts per million, RPM) are indicated in wild-type cells ( D ) or CDK12 knockdown cells ( F ). ( G and H ) Total pairs of complementary 20 to 22-nt vsiRNAs derived from wild-type cells ( G ) or CDK12 knockdown cells ( H ) in each distance category between 5′ and 3′ ends of a complementary vsiRNA pair, shown as −two for pairs with 2-nt overhang at the 3′ end of each strand defined as the canonical vsiRNAs.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Derivative Assay, Knockdown, Infection

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: The proposed model of CDK12 against VSR to maintain host antiviral RNAi immunity. ( A ) In Drosophila cells, dsRNA generated during FHV replication induces host antiviral RNAi immunity, which produces the antiviral effector, vsiRNAs, that results in the degradation of viral RNA and suppression of viral replication. ( B ) FHV encodes B2 protein to counteract antiviral RNAi immunity in multiple ways, including by binding to long dsRNA, interacting with Dcr-2, and binding to siRNA, and B2 is a viral virulence factor necessary for FHV replication. ( C ) CDK12 inhibits the VSR function of B2 and enhances vsiRNAs production, resulting in more potent RNAi immunity against viral infection.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Generated, Binding Assay, Infection

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: CDK12 restricts FHV replication but not FHVΔB2. ( A ) RT‒qPCR analysis of the mRNA levels in S2 cells treated with the indicated dsRNAs. β -gal dsRNA was used in the control. ( B and C ) S2 cells were pretreated with dsRNA against a control (β -gal ) or the indicated genes for 48 h and then transfected with pMT-B2-V5 ( B ) or pMT-GFP ( C ) plasmids for another 48 h. The pretreated cells were infected with FHVΔB2 and the FHV RNA1 levels, relative to the control, were determined by RT‒qPCR at 24 hpi. ( D ) S2 cells were pretreated with dsRNA against a control (β -gal ) or the indicated genes for 48 h and infected with FHV (MOI = 1), prior to viral RNA quantification with RT‒qPCR at 24 hpi. The relative mRNA or viral RNA levels were normalized to the controls ( A–D ). Error bars represent SD in triplicate experiments ( A–D ). Statistical analysis was performed for panels A–D ; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, n.s., not significant (Student’s t -test).

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Control, Transfection, Infection

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: CDK12 induced antiviral RNAi response by inhibiting the function of B2. ( A and B ) S2 cells were pretreated with three different dsRNAs targeting CDK12 or control dsRNA (β -gal ) for 48 h and transfected with pMT-B2-GFP or pMT-GFP plasmids for another 48 h. The GFP-positive cells were sorted by FACS and followed by FHV∆B2 infection. The total RNA was isolated at 24 hpi. The CDK12 mRNA levels ( A ) and FHV RNA1 levels ( B ), relative to the control in GFP group, were determined by RT‒qPCR. ( C ) S2 cells were pretreated with three different dsRNAs targeting CDK12 or control dsRNA (β -gal ) for 48 h and infected with FHV (MOI = 1), prior to viral RNA quantification with RT-qPCR at 24 hpi. ( D ) S2 cells were co-transfected with pMT-B2-GFP and pMT-GFP, pMT-B2-GFP and pMT-V5-CDK12, pMT-GFP and pMT-V5-CDK12, or pMT-GFP for 48 h and infected with FHVΔB2, prior to viral RNA quantification with RT‒qPCR at 24 hpi. ( E ) S2 cells transfected with pMT-V5-CDK12 and pMT-GFP for 48 h and infected with FHV (MOI = 1), prior to FHV RNA quantification with RT-qPCR at 24 hpi. ( F ) S2 cells were pretreated with three different dsRNAs targeting CDK12 or control dsRNA (β -gal ) for 48 h and then co-transfected with psi-CHECK2 plasmids and pMT-B2-GFP or pMT-GFP plasmids for another 48 h. The pretreated cells were treated with dsRNAs against firefly luciferase or β -gal , and both firefly and Renilla luciferase activity were measured at 48 h. The activity ratios between firefly luciferase and Renilla luciferase were shown and normalized to the control in dsRNA targeting β -gal group. Error bars represent SD in three independent experiments performed in triplicate. Statistical analysis was performed using ANOVA; ∗∗∗ P < 0.001, n.s., not significant.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Control, Transfection, Infection, Isolation, Quantitative RT-PCR, Luciferase, Activity Assay

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: The critical amino acids responsible for dsRNA binding and dimerization in B2 were essential for its interaction with CDK12. ( A ) S2 cells co-transfected with pMT-V5-CDK12 plasmid and pMT-B2-GFP or pMT-GFP plasmids were subjected to Co-IP using anti-V5 (left) or anti-GFP (right) and western blot for the immunoprecipitates using the indicated antibodies. Black arrows indicate the target proteins. ( B ) S2 cells transfected with pMT-B2-V5 plasmid for 48 h and infected with FHVΔB2, prior to Co-IP using anti-V5 antibodies, and western blot for the precipitates using anti-CDK12, anti-V5, and anti-GAPDH antibodies. ( C ) Representative images from immunofluorescence staining of co-localization between B2 and CDK12 in S2 cells co-transfected with pMT-V5-CDK12 and pMT-B2-GFP plasmids. ( D ) Lysates from S2 cells co-expressing V5 tagged CDK12 and GFP-tag B2 were untreated or pretreated with RNase A, prior to Co-IP using anti-V5 antibodies. Western blot for the immunoprecipitates using the indicated antibodies. Black arrows indicate the target proteins. ( E ) Schematic representation of B2 containing three α-helices and three mutants deficient in key amino acids for dimerization, dsRNA binding, or Dcr-2 binding functions of B2, respectively. ( F ) S2 cells were co-transfected with pMT-V5-CDK12 plasmid and pMT-B2-GFP or GFP-tagged B2 mutants expressing plasmids, and cells without V5-CDK12 expression were used as a negative control, prior to Co-IP using anti-V5 antibody. The immunoprecipitates were subjected to western blotting using the indicated antibodies.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Infection, Immunofluorescence, Staining, Expressing, Negative Control

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: CDK12 is required for FHV-derived siRNA production. ( A and B ) Length distributions of total small RNAs ( A ) or FHV-derived small RNAs ( B ) sequenced from wild-type or CDK12 knockdown cells infected with FHV (MOI = 1) at 24 hpi. The yellow shade indicated small RNA peaks. ( C and E ) Distributions of each size of FHV-derived small RNA aligned to the positive and negative strands of the FHV genome in wild-type cells ( C ) or CDK12 knockdown cells ( E ). blue, positive-stranded vsiRNAs; red, negative-stranded vsiRNAs. ( D and F ) The distribution of 21 nt-long vsiRNAs reads in the positive and negative-stranded FHV RNA1 and the relative abundances of positive and negative-stranded vsiRNAs (counts per million, RPM) are indicated in wild-type cells ( D ) or CDK12 knockdown cells ( F ). ( G and H ) Total pairs of complementary 20 to 22-nt vsiRNAs derived from wild-type cells ( G ) or CDK12 knockdown cells ( H ) in each distance category between 5′ and 3′ ends of a complementary vsiRNA pair, shown as −two for pairs with 2-nt overhang at the 3′ end of each strand defined as the canonical vsiRNAs.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Derivative Assay, Knockdown, Infection

Journal: mBio

Article Title: CDK12 antagonizes a viral suppressor of RNAi to modulate antiviral RNAi in Drosophila

doi: 10.1128/mbio.02868-24

Figure Lengend Snippet: The proposed model of CDK12 against VSR to maintain host antiviral RNAi immunity. ( A ) In Drosophila cells, dsRNA generated during FHV replication induces host antiviral RNAi immunity, which produces the antiviral effector, vsiRNAs, that results in the degradation of viral RNA and suppression of viral replication. ( B ) FHV encodes B2 protein to counteract antiviral RNAi immunity in multiple ways, including by binding to long dsRNA, interacting with Dcr-2, and binding to siRNA, and B2 is a viral virulence factor necessary for FHV replication. ( C ) CDK12 inhibits the VSR function of B2 and enhances vsiRNAs production, resulting in more potent RNAi immunity against viral infection.

Article Snippet: For western blot, the total protein extracts and the protein from Co-IP assays were separated by 10% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore) at 200 mA for 90 to 120 min. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 Detergent (TBST) at room temperature for 1 h and then incubated with anti-V5 tag antibody (1:10000, Invitrogen, R960-25), anti-GFP (1:4000, Proteintech, 50430–2-AP) antibody, anti-CDK12 (generated by HuaAn Biotechnology Company, Hangzhou, China), or anti-GAPDH (1:20000, Proteintech, 60004) antibody, respectively, at 4°C overnight.

Techniques: Generated, Binding Assay, Infection

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: >Related to . ( A ) Coomassie blue stained gel showing purified CDK12/Cyclin K, CDK13/Cyclin K, GST-CTD, Flag-RPB1 and RNAPII (from pig thymus). ( B ) Coomassie blue stained SDS-PAGE of an in vitro kinase assay using CDK13/Cyclin K and full-length RPB1 for the indicated times. C: control with no ATP added. ( C ) Schematic representation of the domain organization of wild type and truncated constructs used in this study. CTD: C-terminal domain, NTD: N-terminal domain, KiD: kinase domain and RS: arginine and serine-rich domain. ( D ) Coomassie blue stained gel showing purified CDK12/Cyclin K WT or truncated mutant forms as in (C). ( E ) Coomassie blue stained SDS-PAGE of an in vitro kinase assay using CDK12/Cyclin K wild-type (WT) or truncated forms as in (C) on full-length RPB1 for the indicated times in minutes (min). C: control with no ATP added. ( F ) Western blot of in vitro binding reaction, mixing GST-CTD with the indicated forms of CDK12/Cyclin K, followed by binding to glutathione sepharose, and step-wise washes with increasing NaCl concentrations. FT: flow-through.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: Staining, Purification, SDS Page, In Vitro, Kinase Assay, Control, Construct, Mutagenesis, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: ( A ) Schematic representation of the in vitro kinase assay. PP: protein phosphatase. ( B ) Coomassie blue-stained SDS-PAGE gel of the components of the in vitro kinase assay, incubated for the indicated times in minutes (min). Red arrows indicate phosphorylation products throughout the figures of the paper. C: control with no ATP added. ( C ) Western blot of the in vitro kinase assay with CDK9/Cyclin T1 or CDK12/Cyclin K on RNAPII for the indicated times. Ser5P (3E8 antibody), Ser2P (3E10), and Ser2P+Ser5P (S2+S5) (D1G3K) were examined. CDK-specific inhibitors were added for validation (NVP-2 for CDK9; THZ531 for CDK12). C: no ATP; NVP: 2,5 μM NVP-2 added; THZ: 2,5 μM THZ531 added. NTD: N-terminal domain. ( D ) Western blot of the in vitro kinase assay with CDK9/Cyclin T1 or CDK12/Cyclin K on RNAPII followed by dephosphorylation by SSU72/PIN1 for the indicated times. -ATP: no ATP; -SSU72: no SSU72 added. ( E ) Western blot of MRC5 chromatin fractions treated with DRB (100 μM) or THZ531 (1 μM) for the indicated times. Ctr: no drug added. ( F ) Schematic representation of the proposed mechanism for the generation of Ser2P CTD during late elongation. Following Ser2+Ser5 di-phosphorylation by CDK12 or CDK9, Ser5P can be dephosphorylated by SSU72 or other Ser5-specific protein phosphatases (Ser5 PP).

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: In Vitro, Kinase Assay, Staining, SDS Page, Incubation, Control, Western Blot, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: ( A ) Western blot of the Flag IP from HEK293 cells expressing Flag-HA-CDK12. In: Input, Ctr: no Flag-HA-CDK12 expressed, Flag: Flag-HA-CDK12 expressed. ( B ) Schematic of PAF1C and tags used for purification from insect cells. ( C ) Western blot of the in vitro binding reaction, mixing PAF1C and the indicated forms of CDK12/Cyclin K, followed by Strep IP. Ctr: no PAF1C added. ( D ) Western blot of an in vitro kinase assay using CDK12/Cyclin K and RNAPII complemented with PAF1C for the indicated times. Ctr: no PAF1C added, THZ: 2,5 μM THZ531 added. NTD: N-terminal domain, S2+S5: Ser2P + Ser5P. ( E ) Quantification of an in vitro kinase assay using CDK12/Cyclin K and RNAPII comparing the presence and absence of PAF1C in the reaction in (D) (n = 3 independent experiments). Signal is mean with error bars indicating standard error. A.U: arbitrary units. ( F ) Western blot of an in vitro kinase assay using CDK9/Cyclin T1 and RNAPII complemented with PAF1C for the indicated times. As in (D). ( G ) Western blot of an in vitro kinase assay using CDK12/Cyclin K and RNAPII complemented with PAF1C WT or incomplete variants for 5 minutes. ΔATP: no ATP. ( H ) Western blot of an in vitro kinase assay using CDK12/Cyclin K and RNAPII complemented with PAF1C or Flag-CDC73 titrated in for 5 minutes. C: no ATP added, THZ: 2,5 μM THZ531 added. ( I ) Quantification of an in vitro binding assay as in (C). Mean signal from CDK12 or CDK9 (Flag) was normalized to St-CTR9 (negative control) to show the specific binding (n = 3 independent experiments). Signal is mean, with errors bars indicating standard error. NS: not significant and **: p < 0,01 as determined using an unpaired t-test.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: Western Blot, Expressing, Purification, In Vitro, Binding Assay, Kinase Assay, Negative Control

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: Related to . ( A ) Coomassie blue stained gel showing purified PAF1 complex or incomplete PAF1C missing either WDR61, LEO1 or CDC73 subunits ( B ) Western blot of an in vitro binding reaction between the PAF1 complex and CDK9/Cyclin T1 followed by Strep IP. Ctr: no PAF1 complex added. ( C ) Western blot of an in vitro binding reaction between either St-CTR9, PAF1 complex or PAF1CΔCDC73 and CDK12(KiD)/Cyclin K complex followed by Strep IP. Ctr: no PAF1 complex added. ( D ) Western blot of an in vitro binding reaction between either St-CTR9, PAF1 complex or PAF1CΔCDC73 and CDK9/Cyclin T1 complex followed by Strep IP. Ctr: no PAF1 complex added.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: Staining, Purification, Western Blot, In Vitro, Binding Assay

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: ( A ) (Left) AlphaFold2 structural model of CDK12 kinase domain (purple), Cyclin K cyclin fold (yellow) and CDC73-Cyclin K interacting motif (KIM; green). The CDK12 active site indicated. (Right) Zoom-in showing T893 in the CDK12 T-loop in direct contact with Y290 of the CDC73-KIM. ( B ) (Top) Schematic of CDC73 domain organization. NTD: N-terminal domain. (Bottom) Multiple sequence alignment of CDC73-KIM in humans ( Homo sapiens ), zebrafish ( Danio rerio ), fruit fly ( Drosophila melanogaster ) and the worm ( Caenorhabditis elegans ). Fully conserved residues shaded in red. KIM regions important for T-loop opening and the Cyclin K interaction indicated. ( C ) Zoom-in on AF2 structural model of CDK12 (purple)/Cyclin K (yellow)/CDC73-KIM (green and light blue) complex from (A), highlighting in light blue the fully conserved residues as determined in (B). ( D ) Schematic of the CDC73 mutants: CDC73ΔKIM (missing residues 290-324) and CDC73-YY/AA (Y290A, Y293A). ( E ) Western blot of an in vitro kinase assay using CDK12/Cyclin K and RNAPII complemented with PAF1C WT, CDC73 absent (ΔCDC73) or the CDC73 mutant variants shown in (D). ΔATP: no ATP added. NTD: N-terminal domain, S2+S5: Ser2P + Ser5P. ( F ) Quantification of the in vitro binding assay using Strep-CTR9, PAF1C, PAF1CΔKIM or PAF1C(YY) and the CDK12(KiD)/Cyclin K complex. Mean signal from CDK12 (Flag) was normalized to St-CTR9 (negative control) to show the specific binding (n = 3 independent experiments). Signal is mean with error bars indicating standard error. NS: not significant and ***: p < 0,001 as determined using an unpaired t-test.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: Sequencing, Western Blot, In Vitro, Kinase Assay, Mutagenesis, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: Related to . ( A ) AlphaFold2 (AF2) structural model of the trimeric complex of the CDK12 kinase domain, the Cyclin K cyclin fold, and the CDC73-KIM, colored according to the prediction confidence (b-factor, red indicates high confidence). ( B ) Predicted local distance difference test (pLDDT) per position for the five AF2 models of the trimeric complex. pLDDT is > 80. ( C ) Predicted aligned error (PAE) of the five models generated by AF2. This contrasts with other contacts across subunits of the trimer, where predicted aligned error was low across different model predictions. ( D ) Crystal structure model of the yeast CTDK-1 trimer, showing Ctk1 (purple), Ctk2 (yellow) and Ctk3 (green) (PDB accession code: 7JV7). ( E ) Cryo-EM model of the RNAPII elongation complex containing DSIF (dark blue), SPT6 (light blue) and the PAF1 complex (green) (PDB accession code: 6TED). The CDC73 C-terminus (pink) was modelled onto the SPT6 C-terminal SH2 domain using AF2 and superimposed. CDK12/Cyclin K/CDC73-KIM was then added to the model in an approximate location in the vicinity of RNAPII CTD. ( F ) Coomassie blue stained gel showing purified PAF1 complex, PAF1CΔKIM and PAF1C-YY/AA. ( G ) Western blot of an in vitro binding reaction between either St-CTR9, PAF1 complex, PAF1CΔKIM or PAF1C-YY/AA and CDK12(KiD)/Cyclin K complex followed by Strep IP. Ctr: no PAF1 complex added.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: Generated, Cryo-EM Sample Prep, Staining, Purification, Western Blot, In Vitro, Binding Assay

Journal: bioRxiv

Article Title: PAF1C allosterically activates CDK12/13 kinase during RNAPII transcript elongation

doi: 10.1101/2024.10.14.618141

Figure Lengend Snippet: ( A ) Schematic of the switchover system. Following knock-down of endogenous CDC73, expression of the recombinant, siRNA resistant, Flag tagged CDC73 (Flag-CDC73*) is induced with doxycycline (Dox; 1µg/mL) for 24 hours (h). siCDC73: siRNA targeting CDC73. siCon: siRNA targeting control locus. ( B ) Cell confluence (in (%)) measured over time using the switchover system. Cell confluence was tracked every 4 h for 5 days. Point indicates mean of technical duplicate and error bars indicate standard deviation. Image is representative of experiment performed in biological triplicate. F: Flag. ( C ) Western blot of chromatin fractions following switchover, measuring total RPB1, Ser2P, Ser5P and Ser2P-Ser5P signals. ( D ) Representative images of nascent RNA read counts across SETX and EXT1 , with the direction of transcription indicated by arrow. ( E ) Metagene analysis of non-overlapping coding genes longer than 100 kb (n = 2217) using library- and spike-in normalized nascent RNA read counts obtained from transient transcriptome chem sequencing (TTchem-seq). Line indicates mean signal of experiment performed in technical triplicate. Shaded area indicates standard error. Si73: siRNA targeting CDC73, CPM: counts per million, TSS: transcription start site, TES: transcription end site, kb: kilobases. ( F ) Same as (E) except metagene analysis was performed using non-overlapping coding gene intervals longer than 50 kb (n = 3860) showing the TSS and first 20 kb. ( G ) Schematic of PAF1C-mediated CDK12 activation during transcript elongation.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), alpha Tubulin (ab52866, Abcam) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4 °C.

Techniques: Knockdown, Expressing, Recombinant, Control, Standard Deviation, Western Blot, Sequencing, Activation Assay