Journal: bioRxiv

Article Title: TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth

doi: 10.1101/2022.02.20.481212

Figure Lengend Snippet: (A) BRF1 and TIF-1A were co-localized in the nucleoli of HeLa cells. IF staining was performed using the BRF1 and TIF-1A antibodies. The scale bar in each image represents 2.5 μm. ( B ) BRF1 and RPA40 were co-localized in the nucleoli of HeLa cells. IF staining was performed using BRF1 antibody and RPA40 antibody. The scale bar in each image represents 2.5 μm. ( C ) The co-localization analysis between BRF1 and TIF-1A using the nucleolar particles purified from HeLa cells. The scale bar in each image represents 2.5 μm. ( D ) The co-localization analysis between BRF1 and RPA43 using the nucleoli particles purified from HeLa cells. The scale bar in each image represents 5 μm. ( E ) Components of the Pol I transcription machinery could be precipitated by the BRF1 antibody. HeLa nuclear extract was prepared and used for BRF1 IP assays. BRF1-binding proteins in the IP samples were detected by Western blot using the antibodies as indicated in the panel. ( F , G ) BRF1 could be precipitated by the antibodies against Pol I-related proteins. IP assays were performed with the antibodies against TBP, TIF-1A, TAF1A, UBF, and RPA43, respectively. BRF1 ( F ) and proteins as indicated ( G ) were detected by Western blot. Figure 6-figure supplement 1. The co-localization analysis between BRF1 and Pol III transcription factors in HeLa cells. Figure 6-figure supplement 2. BRF1 binds to TFIIIC subunit GTF3C2 and SL1 subunit TAF1B. Figure 6-source data, Figure 6-figure supplement 2-source data

Article Snippet: IP assays were performed using normal IgG, BRF1 antibody and the antibodies against UBF (ab244287, Abcam), TIF-1A (ab251933, Abcam), TBP (ab28175, abcam), TAF1A (SC-393600, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), TAF1B (CSB-PA684476ESR1HU, CUSABio) and RPA43 (Ab99305, Abcam), respectively.

Techniques: Staining, Purification, Binding Assay, Western Blot

Journal: bioRxiv

Article Title: TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth

doi: 10.1101/2022.02.20.481212

Figure Lengend Snippet: (A) BRF1 IP results showing BRF1 association with GTF3C2 and TAF1B. IP assays were performed using BRF1 antibody and HeLa nuclear extract. BRF1-bound protein was detected by Western blot using the antibodies as indicated. ( B ) GTF3C2 IP result showing GT3C2 association with BRF1. IP assays were performed using GTF3C2 antibody and HeLa nuclear extract. GTF3C2-bound protein was detected by Western blot using the antibodies as indicated. ( C ) TAF1B IP result showing TAF1B association with BRF1. IP assays were performed using TAF1B antibody and HeLa nuclear extract. TAF1B-bound protein was detected by Western blot using the antibodies as indicated.

Article Snippet: IP assays were performed using normal IgG, BRF1 antibody and the antibodies against UBF (ab244287, Abcam), TIF-1A (ab251933, Abcam), TBP (ab28175, abcam), TAF1A (SC-393600, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), TAF1B (CSB-PA684476ESR1HU, CUSABio) and RPA43 (Ab99305, Abcam), respectively.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Derivative Assay, Sequencing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Schematic representation showing the relative positions of Cas9 target sites for small guide RNAs sg-1 to sg-3. (B) Akata (EBV+) cells were used to establish stable cell lines using 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated cross-linking of BCR. YTHDF2 and viral protein (ZTA and RTA) expression levels were monitored by Western Blot using antibodies as indicated. (C) RNAs from YTHDF2-depleted and control Akata cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (D) P3HR-1 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with TPA and sodium butyrate (NaBu) to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (E) RNAs from YTHDF2-depleted and control P3HR-1 cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (F) SUN-719 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with Gemcitabine to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (G) Akata (EBV+) cells were used to establish control and YTHDF2 overexpression cell line as indicated. The cells were untreated or lytically induced by anti-IgG treatment. The expression of YTHDF2 as monitored by anti-YTHDF2 and anti-Myc antibodies. Viral protein expression levels were monitored by Western Blot using antibodies as indicated. (H) Extracellular virion-associated DNA from cells treated in panel G was extracted and the relative EBV viral copy numbers were calculated by q-PCR analysis using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p<0.001. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2 or 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG treatment for 24 or 48 hrs as indicated. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (A) EIF4H depletion promotes the expression of EBV ZTA and RTA. (B) MAGEA10 depletion does not affect EBV protein expression. (C) SORT1 depletion does not significantly affect EBV protein expression. (D) EHMT2 depletion does not affect EBV protein expression. Arrowhead denotes cleaved fragments. (E) MTA1 depletion does not uniformly affect EBV protein expression but slightly enhances the expression of its homolog MTA2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) YTHDF2-depleted and control Akata (EBV+) cells were lytically induced with anti-IgG for 0 to 48 hrs. (B) YTHDF2-depleted and control P3HR1 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. (C) YTHDF2-depleted and control SNU-719 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, -8 and -6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of caspases, cleaved caspases, YTHDF2, PIAS1 and viral proteins (ZTA and RTA) was monitored by Western Blot using antibodies as indicated. Arrowheads denote cleaved fragments. See also - and Table S2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Functional Assay, Labeling, Binding Assay, Modification, Incubation, Recombinant, Mutagenesis, Generated

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-C) Akata (EBV+) cells were lytically induced with anti-IgG for 0, 6, 12, 24 and 48 hrs (A). P3HR1 (B) and SNU-719 (C) cells were lytically induced with TPA and NaBu for 0 6, 12, 24 and 48 hrs. YTHDF2, EBV ZTA and RTA, cleaved caspase substrate (CASP sub.), cleaved PARP, cleaved CASP3 and cleaved CASP8 were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (D-F) Apoptotic induction by an intrinsic trigger promotes EBV reactivation. Akata (EBV+) (D), P3HR1 (E) and SNU-719 (F) cells were untreated or treated with increasing amount of Taxol for 48 hrs. YTHDF2, EBV ZTA and RTA, and cleaved caspase substrate (CASP sub.) were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata EBV(+) cells were transduced with lenti-vector control or Myc-CASP8 to establish stable cell lines. The cells were treated with anti-IgG for 0, 24 and 48 hrs. (A) CASP8, EBV ZTA and RTA were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (B) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. (C) Total RNA was extracted and then EBV lytic (ZTA and RTA) and latent (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 and LMP2) genes were analyzed by RT-qPCR. The value of vector control at 0 hr was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. (A-B) Total RNA was extracted and then EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. (C) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic Akata (EBV+) cells upon lytic induction. Akata (EBV+) cells were either untreated (control) or treated with anti-IgG antibody for 24 and 48 hrs as indicated. (A) The cells were stained with Propidium Iodide (PI) and then permeabilized for immnunostaining with anti-YTHDF2 antibody. (B) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. (C-D) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic SNU-719 cells upon lytic induction. SNU-719 cells were either untreated (control) or treated with TPA and NaBu for 24 and 48 hrs as indicated. (C) Cells were stained with Propidium Iodide (PI) and then permeabilized for immunostaining with anti-YTHDF2 antibody. (D) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. Scale bar, 20 μm

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The Akata (EBV+) (A), P3HR-1 (B) and SNU-719 (C) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then lytically induced with anti-IgG antibody or TPA/NaBu as indicated for 48 hrs. Western Blot showing the protein levels of RIP, phospho-RIP (p-RIP) and phospho-RIP3 (p-RIP3) using antibodies as indicated. β-actin blots were included as controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The design of CRISPR/Cas9-resistant YTHDF2 variant was based on the sg-2 protospacer adjacent motif (PAM). D166A/D367A mutations were introduced into the PAM-mutated YTHDF2. Both constructs were cloned into a lentiviral vector with a C-terminal Myc-tag. (B-C) WT and cleavage-resistant YTHDF2 suppresses EBV replication. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing YTHDF2 and EBV protein expression levels in these cell lines upon IgG cross-linking as indicated (B). Arrowheads denote cleaved fragments. Extracellular and intracellular viral DNA was measured by qPCR using primers specific to BALF5 (C). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001. (D) Schematic representation of 5 YTHDF2 cleavage-mimicking fragments. These fragments were cloned into a lentiviral vector with a C-terminal Myc-tag. (E) SNU-719 cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. (F) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (G) Caspase-mediated cleavage impairs YTHDF2 binding to CNOT1. Halo-V5-tagged WT YTHDF2 and the individual fragments were co-transfected with HA-tagged CNOT1 SH domain into 293T cells as indicated. Co-immunoprecipitation (Co-IP) experiments were performed using anti-V5 antibody-conjugated magnetic beads. The immunoprecipitated samples and total cell lysates (Input) were analyzed by Western Blot with antibodies as indicated. (H) Model showing the functional consequences of YTHDF2 cleavage in CNOT1 binding and the targeting of m 6 A-modified RNA. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: CRISPR, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Transduction, Stable Transfection, Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Functional Assay, Modification

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) SNU-719 cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines (see ). RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. The value of vector control was set as 1. (B) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines . RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. The value of vector control at 24 hrs was set as 1. (C) Akata (EBV+) cells were transduced with lentiviruses carrying vector control, Halo-tag, WT YTHDF2 or individual YTHDF2 fragment to establish stable cell lines. Western Blot analysis showing Halo, YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (D) Total mRNA was extracted from cells treated in panel (C). EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. The value of vector control at 24 hrs was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. N.S., not significant; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were lytically induced by IgG-cross linking for 24 hrs. (A) Total RNA was subjected to m 6 A RIP, followed by RT-qPCR using indicated primers. Values are displayed as fold change over 10% input. GAPDH and Dicer are cellular negative and positive controls, respectively. (B) Cell lysate was collected to detect YTHDF2 binding of viral RNAs by RIP-qPCR. Values are displayed as fold change over 10% input. MALAT1 and SON are cellular negative and positive controls, respectively. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( , , ) (B-C) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated (B). CASP8 mRNA levels were analyzed by RT-qPCR using CASP8 primers (C). The value of vector control at 0 hr was set as 1. (D-E) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (D). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (E). The value of vector control at 0 hr was set as 1. (F-G) Caspase-8 depletion suppresses EBV replication in YTHDF2-depleted cells. YTHDF2-depleted Akata (EBV+) cells were transduced with lentivirus carrying control sgRNA or CASP8-sg1 to establish cell lines and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (F). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (G). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Activation Assay, Activity Assay, Derivative Assay, Expressing, Construct, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Inhibition, Transduction

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic induction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.. The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001. See also , and Table S3.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Expressing, Activation Assay, Modification, Binding Assay, Western Blot, Derivative Assay, Generated, Mutagenesis, Clone Assay, Luciferase, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Modification, Western Blot, Inhibition, Incubation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Incubation, Western Blot, Recombinant, Sequencing, Generated, Stable Transfection, Construct, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A), METTL14 (B), WTAP (C), VIRMA (D) and YTHDF3 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A and B), METTL14 (C and D), WTAP (E and F), VIRMA (G and H) and YTHDF3 (I and J) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation for 24 or 48 hrs. EBV ZTA and RTA mRNA expression levels were monitored by RT-qPCR (A, C, E, G and I). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (B, D, F, H and J). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation