Journal: Cell reports

Article Title: Kaposi’s sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis

doi: 10.1016/j.celrep.2021.110144

Figure Lengend Snippet: (A) Cap-binding proteins pulled down with m 7 GTP agarose-beads from un-infected, latent, and reactivated iSLKs. (B) m 7 GTP pulled-down viral and host proteins in the presence or absence of RNase, 24 h post-reactivation. (C) eIF4E2-HA immunoprecipitation (IP) in 48 h reactivated iSLK.KSHV219. (D) eIF4E1-HA-associated proteins 48 h post-reactivation. (E) HIF2α-HA IP 24 h post-reactivation. (F) Polysome profiles of siControl and sieIF4E2 iSLK.KSHV219 cells 48 h post-reactivation. Silenced cells 48 h post-DOX were treated with cycloheximide (CHX), and lysates were sedimented through sucrose gradients and fractionated, and viral mRNAs from each fraction were detected using qRT-PCR. Ribosome subunits (40S, 60S), monosomes (80S), oligosomes, and polysomes are indicated. The table shows the area under the curve (AOC) of each fraction. (G) Translation efficiency of KSHV lytic mRNAs in cells from (F). KSHV mRNA levels in all fractions were measured using qRT-PCR (n = 3). Each fraction CT value was normalized to the 80S fraction CT. (H) Input mRNA levels of KSHV lytic genes prior polysome profiling measured using qRT-PCR 48 h post-DOX relative to siControl (n = 3; mean ± SD; *p < 0.0001, two-way ANOVA with Sidak’s post-test). (I) Translation efficiency of eIF4E1-dependent host gene RPL3 in cells from (F). RPL3 mRNA levels in all fractions were measured as in (G). (J) Fold enrichment of KSHV mRNAs after endogenous HIF2α RNA immunoprecipitation (RIP) relative to IgG RIP control at 24 h post-DOX. KSHV mRNA levels were quantified using qRT-PCR, and CT values were first normalized to input CT (n = 3; mean ± SD; *p < 0.05, two-way ANOVA with Sidak’s post-test). (K) Pull-down levels of KSHV mRNAs after eIF4E2-HA and eIF4E1-HA RIP relative to empty control at 24 h post-DOX. KSHV mRNA levels were quantified as in (J) (n = 3; mean ± SD; *p < 0.05, two-way ANOVA with Tukey’s post-test). (L) Host gene pull-down levels in cells from (K) (n = 3; mean ± SD; ns, not significant, two-way ANOVA with Tukey’s post-test).

Article Snippet: m 7 GTP agarose beads , Jena Biosciences , Cat.# AC-155S.

Techniques: Binding Assay, Infection, Immunoprecipitation, Quantitative RT-PCR

Journal: Cell reports

Article Title: Kaposi’s sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis

doi: 10.1016/j.celrep.2021.110144

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: m 7 GTP agarose beads , Jena Biosciences , Cat.# AC-155S.

Techniques: Immunohistochemistry, Recombinant, Lysis, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Amplification, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Software, Real-time Polymerase Chain Reaction, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Driver mutations of the adenoma-carcinoma sequence govern the intestinal epithelial global translational capacity

doi: 10.1073/pnas.1912772117

Figure Lengend Snippet: Knockdown of Smad4 results in enrichment of the stem and Paneth cell compartments, which is accompanied by increased global translation and reduced expression of translational repressor 4E-BP1. (A) Quantitative RT-PCR analysis of crypt base columnar stem cell markers in shSmad4 organoids (n = 3). (B) Analysis of small intestinal differentiation markers in shSmad4 organoids (n = 3). (C) Combined staining of in situ hybridization of stem cell marker lgr5 and immunostaining for Paneth cell marker lysozyme in shSmad4 organoids, including quantification of the mRNA particles and lysozyme-positive cells per organoid. *P < 0.05, Student’s t test. (D) l-[35S]-methionine incorporation assay of shSmad4 organoids assessed at day 4 after passaging (n = 3). (E) Clonogenic capacity of single cells that grow out to fully developed organoids, presented as a quantification of the organoid number per 20,000 seeded cells (n = 3). (F) l-[35S]-methionine incorporation assay in Apc−/− Kras+/G12D shSmad4#2 (AKS) and Apc−/− Kras+/G12D shControl (AK) organoids assessed at day 3 after passaging (n = 4). (G) Representative immunoblotting analysis of 4E-BP1 expression in shSmad4 organoids. β-Actin served as a loading control (n = 3). (H) Representative immunoblotting analysis of phospho-4E-BP1 (Thr70), eIF4E, and eIF4G in shSmad4 organoids. Optical density ratios of phospho-4E-BP1/4E-BP1 and 4E-BP1/eIF4E were also calculated (n = 2). (I) m7GTP-agarose pulldown assay in shSmad4 organoids to assess the levels of cap-bound eIF4E, eIF4G, and 4E-BP1 (n = 3). Unbound levels of β-actin 4E-BP1 and eIF4G were detected in supernatants. (J) m7GTP-agarose pulldown assay comparing AKS (shSmad4 #2) to AK organoids (shControl) (n = 3). Data are represented as means ± SEM. Significance (one-way ANOVA) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: 30 to 50 µL of 7-methyl-GTP-agarose beads (Immobilized γ-Aminophenyl-m7GTP-C10-spacer, Jena Bioscience) was added and samples were incubated overnight.

Techniques: Expressing, Quantitative RT-PCR, Staining, In Situ Hybridization, Marker, Immunostaining, Passaging, Western Blot

Journal: bioRxiv

Article Title: IFIT3 Associates with m⁶A-Modified RNA to Restrict Hepatitis C Virus Infection

doi: 10.64898/2026.03.25.714224

Figure Lengend Snippet: ( A ) Schematic of biotinylated RNA probe +/- m⁶A used for cell-free RNA binding assays. ( B ) Representative immunoblot and corresponding quantitation across replicates of IFIT3-V5 from IFNβ-treated (100 U/ml, 24 h) 293T cytoplasmic extracts bound to synthetic biotinylated RNA probes after streptavidin-bead purification and washing. Lanes represent independent RNA affinity pulldown assays where +/- m⁶A probes were incubated with extracts. ( C-D ) Electrophoretic mobility shift assay of IFIT3 and YTHDC1 with RNA probe (as in A with no 5’ biotin) ± m 6 A modification. For all gels, protein concentrations in each well are: 0, 0.065, 0.130, 0.260, 0.521, 1.04 and 2.08 μM (left to right). ( E ) Diagram of DRACH motif mutations introduced into HCV genomic RNA in proximity to the 2098 IFIT3-ADAR and YTH-ADAR editing sites (indicated as cyan dot). Previous SHAPE-seq was used to depict secondary structure. Published YTHDF PAR-CLIP peaks are indicated in purple. Yellow and red dots represent DRACH motifs with red dots indicating putative m 6 A nucleotides. Nucleotide mutations are indicated at respective DRACH motifs with codons separated by “|”. ( F ) Diagram of DRACH motif mutations introduced into HCV genomic RNA in proximity to the 3131 IFIT3-ADAR and YTH-ADAR editing sites (indicated as cyan dot). Annotations are as explained in (E). ( G ) RT-PCR Sanger sequencing quantification of IFIT3-ADAR and YTH-ADAR A:I editing at nt 2098 on WT or mutant HCV RNA 72 h post electroporation. Values are normalized to WT editing. ( H ) RT-PCR Sanger sequencing quantification of IFIT3-ADAR and YTH-ADAR A:I editing at nt 3131 on WT or mutant HCV RNA 72 h post electroporation. Values are normalized to WT editing. See also Figure S3.

Article Snippet: For RNA pulldowns, synthesized 38-nt biotinylated RNA probes (Horizon; 5’-biotin-GGGGCGUGUGGUCGGG[A/m6A]CUCGGCUUGGCUGCGCGUCCC-3’) were denatured at 95°C for 5 min, snap-cooled on ice, and immobilized on pre-washed streptavidin magnetic beads (250 pmol RNA per 5 μL beads) in 200 μL binding buffer (10 mM Tris-Cl pH 7.5, 1.5 mM MgCl2, 150 mM KCl, 0.05% NP-40, protease inhibitor (Sigma-Aldrich), 0.5 mM DTT, 1 × 105 U/mL murine RNase inhibitor (NEB-) for 2 h at 4°C with rotation.

Techniques: RNA Binding Assay, Western Blot, Quantitation Assay, Purification, Incubation, Electrophoretic Mobility Shift Assay, Modification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Mutagenesis, Electroporation

Journal: bioRxiv

Article Title: IFIT3 Associates with m⁶A-Modified RNA to Restrict Hepatitis C Virus Infection

doi: 10.64898/2026.03.25.714224

Figure Lengend Snippet: ( A ) Diagram of human IFIT3 domain deletions used for assays B-I. ( B-C ) Representative immunoblot and corresponding quantitation across replicates of transfected IFIT3-V5 WT or mutant (see 4A) from IFNβ-treated (100 U/ml, 24 h) IFIT3 knockout 293T cytoplasmic extracts bound to synthetic biotinylated RNA probes after streptavidin-bead purification and washing. Lanes represent independent RNA affinity pulldown assays where m⁶A probes (see 3A) were incubated with extracts. ( D ) Predicted 3d structure of human IFIT3 (UniProt: O14879, Alphafold3 server ) with TPRs, putative IFIT2 interaction “swap domain” , and target regions for deletion annotated. ( E ) Immunoblotting of IFIT3-V5 WT or mutant (see 4A) co-precipitation with Flag-IFIT1 co-expressed in IFNβ-treated (100 U/ml, 24 h) IFIT1-IFIT3 double-knockout 293T cells. ( F ) Immunoblotting of IFIT3-V5 WT or mutant (see 4A) co-precipitation with HA-IFIT2 co-expressed in IFNβ-treated (100 U/ml, 24 h) IFIT1-IFIT2 double-knockout 293T cells. ( G ) Immunoblotting of IFIT3-V5 WT or mutant (see 4D) co-precipitation with Flag-IFIT1 co-expressed in IFNβ-treated (100 U/ml, 24 h) IFIT1-IFIT3 double-knockout 293T cells. ( H ) Immunoblotting of IFIT3-V5 WT or mutant (see 4D) co-precipitation with HA-IFIT2 co-expressed in IFNβ-treated (100 U/ml, 24 h) IFIT1-IFIT2 double-knockout 293T cells. ( I ) Representative Immunoblotting of expression and immunoprecipitation of stably expressed WT or mutant IFIT3-V5 in HCV-infected (MOI 1, 24 hpi) IFIT1-IFIT3 double-knockout Huh7 cells for native RIP-qPCR (left). Native RIP-qPCR of co-purified HCV RNA from immunoprecipitated fractions shown in. Values represent enrichment of V5-purified RNA in comparison to IgG followed by normalization to WT IFIT3 (right).

Article Snippet: For RNA pulldowns, synthesized 38-nt biotinylated RNA probes (Horizon; 5’-biotin-GGGGCGUGUGGUCGGG[A/m6A]CUCGGCUUGGCUGCGCGUCCC-3’) were denatured at 95°C for 5 min, snap-cooled on ice, and immobilized on pre-washed streptavidin magnetic beads (250 pmol RNA per 5 μL beads) in 200 μL binding buffer (10 mM Tris-Cl pH 7.5, 1.5 mM MgCl2, 150 mM KCl, 0.05% NP-40, protease inhibitor (Sigma-Aldrich), 0.5 mM DTT, 1 × 105 U/mL murine RNase inhibitor (NEB-) for 2 h at 4°C with rotation.

Techniques: Western Blot, Quantitation Assay, Transfection, Mutagenesis, Knock-Out, Purification, Incubation, Double Knockout, Expressing, Immunoprecipitation, Stable Transfection, Infection, Comparison