Journal: FEBS Open Bio

Article Title: Characterization of WAC interactions with R2TP and TTT chaperone complexes linking glucose and glutamine availability to mTORC1 activity

doi: 10.1002/2211-5463.70085

Figure Lengend Snippet: WAC interacts with the R2TP complex. (A) Pull‐down experiment testing the interaction between WAC‐3xFlag and the R2TP complex. WAC‐3xFlag, used as bait, was incubated with a purified, already assembled R2TP complex. (B) To map the interaction of WAC with the subunits of the R2TP complex, a similar experiment as in (A) was performed to test the interaction with the RUVBL1‐RUVBL2 complex. Similar experiments are shown but using RPAP3 (C) and RPAP3‐PIH1D1 (D) as prey. Assays were performed as explained in the Methods section, similarly to the experiments shown in Fig. .

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti‐FLAG M2 antibody (Sigma‐Aldrich, F1804, 1 : 2000), HA (Abcam #1091591; 1 : 1000), mLST8_GBL 86B8 (Cell Signaling #3274; 1 : 1000), mTOR (7C10, Cell signaling #2972; 1 : 1000), RagA/B D8B5 (Cell Signaling #4357; 1 : 1000), RAPTOR (24C12) (Cell signaling, #2280; 1 : 1000), S6 ribosomal protein (Cell Signaling #2217; 1 : 1000), p‐S6 ribosomal protein (Ser240/244) (Cell Signaling #2215; 1 : 1000), TELO2 (15975‐1‐AP, Proteintech, Rosemont, IL, USA; 1 : 1000), TTI1 (A303‐451A, Bionova Scientific; 1 : 1000), TTI2 (A303‐476A, Bionova Scientific, Fremont, CA, USA; 1 : 500), RPAP3 (Invitrogen #PA5‐58335; 1 : 500), RUVBL1 (Cell signaling #12300; 1 : 500), RUVBL2 (Cell signaling #8959; 1 : 500), PIH1D1 (Invitrogen PA5‐61482, 1 : 500).

Techniques: Incubation, Purification

Journal: FEBS Open Bio

Article Title: Characterization of WAC interactions with R2TP and TTT chaperone complexes linking glucose and glutamine availability to mTORC1 activity

doi: 10.1002/2211-5463.70085

Figure Lengend Snippet: Analysis of gene and protein levels for WAC, mTORC1, R2TP, and TTT across cancer types. Gene (A) and protein (B) levels of the subunits of the mTOR‐WAC‐RUVBL1‐RUVBL2‐TTT complex in 10 cancer types with CPTAC tumor samples from LinkedOmics. Z‐scores are shown to represent normalized expression levels, with red indicating relatively high expression and blue indicating relatively low expression. The number of cancer types showing a positive correlation in gene (C) and protein (D) levels between subunits of the mTOR‐WAC‐RUVBL1‐RUVBL2‐TTT complex across 10 cancer types. The number within each box indicates the number of cancer types with a positive correlation out of the 10 tested. A positive correlation is defined as a Pearson correlation coefficient > 0.2 with a P ‐value < 0.01. (E) Distribution of WAC protein expression in tumor samples versus matched normal samples across cancer types (where normal samples are available). Statistically significant differences between tumor and normal conditions were assessed using the two‐sided Wilcoxon rank sum test. Units in the Y axis correspond to protein expression levels in LinkedOmics, provided as log₂‐transformed MS1 intensities, which represent relative protein abundances measured via mass spectrometry and normalized for comparative analyses. (F) Differences in protein levels of the components of the WAC‐RUVBL1‐RUVBL2‐TTT complex between tumor and adjacent normal tissue (NAT) across cancer types. Red indicates higher expression in tumors compared to normal samples, while blue indicates higher expression in normal samples compared to tumors. Statistically significant differences were assessed using the two‐sided Wilcoxon rank sum test. The meta P ‐value represents pan‐cancer significance when all samples are analyzed together using the two‐sided Wilcoxon rank sum test. * P < 0.05, ** P < 0.01, *** P < 0.001. BRCA, breast invasive carcinoma; CCRCC, clear cell renal cell carcinoma; COAD, colon adenocarcinoma; GBM, glioblastoma; HNSCC, head and neck squamous cell carcinoma; LSCC, lung squamous cell carcinoma; LUAD, lung adenocarcinoma; OV, ovarian serous cystadenocarcinoma; PDAC, pancreatic ductal adenocarcinoma; UCEC, uterine corpus endometrial carcinoma.

Article Snippet: Primary antibodies used in western blotting with dilutions were as follows: monoclonal Anti‐FLAG M2 antibody (Sigma‐Aldrich, F1804, 1 : 2000), HA (Abcam #1091591; 1 : 1000), mLST8_GBL 86B8 (Cell Signaling #3274; 1 : 1000), mTOR (7C10, Cell signaling #2972; 1 : 1000), RagA/B D8B5 (Cell Signaling #4357; 1 : 1000), RAPTOR (24C12) (Cell signaling, #2280; 1 : 1000), S6 ribosomal protein (Cell Signaling #2217; 1 : 1000), p‐S6 ribosomal protein (Ser240/244) (Cell Signaling #2215; 1 : 1000), TELO2 (15975‐1‐AP, Proteintech, Rosemont, IL, USA; 1 : 1000), TTI1 (A303‐451A, Bionova Scientific; 1 : 1000), TTI2 (A303‐476A, Bionova Scientific, Fremont, CA, USA; 1 : 500), RPAP3 (Invitrogen #PA5‐58335; 1 : 500), RUVBL1 (Cell signaling #12300; 1 : 500), RUVBL2 (Cell signaling #8959; 1 : 500), PIH1D1 (Invitrogen PA5‐61482, 1 : 500).

Techniques: Expressing, Transformation Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Influenza A virus polymerase co-opts distinct sets of host proteins for RNA transcription or replication

doi: 10.1101/2025.06.06.658254

Figure Lengend Snippet: HMGB2, RUVBL2, and RPAP2 promote IAV polymerase activity. A) Minigenome reporter assay in eHAP WT cells transfected with a non-targeting siRNA (siNT), two siRNA pools targeting both ANP32A and ANP32B (siANP32.1 and siANP32.2), or siRNA pools targeting 31 genes enriched in WT cells or 2 genes enriched in both WT and ANP32-KO cells. Cells were transfected with plasmids to reconstitute PR8 polymerase activity. Firefly luciferase activity was normalized to co-transfected Renilla luciferase levels. Putative replication-specific proviral factors are shown in green, replication-specific antiviral factors in red, and transcription-specific proviral factors in blue. Statistical significance was assessed compared to siNT using an unpaired t test. B-I) Minigenome reporter assay in A549 WT and HMGB2-KO cells (B-E) or WT and RUVBL2-KO cells (F-I) transfected with plasmids to reconstitute PR8 (B and F), Vic75 (C and G), Eng195 (D and H), and Tky05 (E and I) polymerase activity. Firefly luciferase activity was normalized to co-transfected Renilla luciferase levels. Statistical significance was assessed compared to WT using an unpaired t test. J-O) Accumulation of segment 4 (Haemagglutinin (HA)) vRNA (J and M), cRNA (K and N), and mRNA (L and O) in A549 WT and RPAP2-KO cells (J-L) or WT and HMGB2-KO cells (M-O) following infection with PR8 (MOI=3). Data is shown as the fold change over input (0hpi). Statistical significance was assessed at each timepoint following log transformation using multiple unpaired t tests, corrected for multiple comparisons using the FDR. For all panels: Data is shown as mean of n=3 technical repeats, representative of n=3 biological repeats. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. See also Figure S4 and S5.

Article Snippet: Commercial multi-guide sgRNAs targeting HMGB2, RUVBL2, and RPAP2 (Synthego) and non-targeting sgRNA (Synthego) were used.

Techniques: Activity Assay, Reporter Assay, Transfection, Luciferase, Infection, Transformation Assay

Journal: bioRxiv

Article Title: Influenza A virus polymerase co-opts distinct sets of host proteins for RNA transcription or replication

doi: 10.1101/2025.06.06.658254

Figure Lengend Snippet: HMGB2 and RUVBL2 are replication-specific cofactors and RPAP2 is a transcription-specific cofactor. A) Schematic showing vRNA, cRNA, and mRNA synthesis in a minigenome setup with a WT, replication-deficient (PA E410A), or transcription-deficient (PA D108A) polymerase. B and C) cRNA:vRNA ratio (B) and mRNA:vRNA ratio (C) produced by a PR8 WT vRNP following transient knockdown of both ANP32A and ANP32B (siANP32.1 and siANP32.2), HMGB2, RUVBL2, or RPAP2. Activity of a replication-deficient vRNP (green striped) and transcription-deficient vRNP (blue striped) in cells transfected with a non-targeting siRNA (siNT) is also shown. cRNA:vRNA ratios were normalized to the ratio produced by the transcription-deficient vRNP in siNT-treated cells (B). mRNA:vRNA ratios were normalized to the ratio produced by the replication-deficient vRNP in siNT-treated cells (C). vRNA template provided was a pPolI-Firefly luciferase plasmid. Statistical significance was assessed compared to replication-deficient/transcription-deficient polymerases using an unpaired t test. D) Schematic showing vRNA, cRNA, and mRNA synthesis under conditions of a cRNP stabilization assay with cycloheximide (CHX) treatment. E-H) cRNP stabilization assay with CHX in A549 WT and HMGB2-KO cells (E and F) or WT and RPAP2-KO cells (G and H). Data is shown as fold change in segment 4 (Haemagglutinin (HA)) cRNA:vRNA ratio (E and G) and mRNA:vRNA ratio (F and H) over input (0hpi). Statistical significance was determined at each timepoint following log transformation using multiple unpaired t tests, corrected for multiple comparisons using the FDR. For (B), (C), and (E-H): Data is shown as mean of n=3 technical repeats, representative of n=3 biological repeats. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figure S6.

Article Snippet: Commercial multi-guide sgRNAs targeting HMGB2, RUVBL2, and RPAP2 (Synthego) and non-targeting sgRNA (Synthego) were used.

Techniques: Produced, Knockdown, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Transformation Assay

Journal: bioRxiv

Article Title: Influenza A virus polymerase co-opts distinct sets of host proteins for RNA transcription or replication

doi: 10.1101/2025.06.06.658254

Figure Lengend Snippet: HMGB2, RUVBL2, and RPAP2 promote IAV polymerase activity. A) Minigenome reporter assay in eHAP WT cells transfected with a non-targeting siRNA (siNT), two siRNA pools targeting both ANP32A and ANP32B (siANP32.1 and siANP32.2), or siRNA pools targeting 31 genes enriched in WT cells or 2 genes enriched in both WT and ANP32-KO cells. Cells were transfected with plasmids to reconstitute PR8 polymerase activity. Firefly luciferase activity was normalized to co-transfected Renilla luciferase levels. Putative replication-specific proviral factors are shown in green, replication-specific antiviral factors in red, and transcription-specific proviral factors in blue. Statistical significance was assessed compared to siNT using an unpaired t test. B-I) Minigenome reporter assay in A549 WT and HMGB2-KO cells (B-E) or WT and RUVBL2-KO cells (F-I) transfected with plasmids to reconstitute PR8 (B and F), Vic75 (C and G), Eng195 (D and H), and Tky05 (E and I) polymerase activity. Firefly luciferase activity was normalized to co-transfected Renilla luciferase levels. Statistical significance was assessed compared to WT using an unpaired t test. J-O) Accumulation of segment 4 (Haemagglutinin (HA)) vRNA (J and M), cRNA (K and N), and mRNA (L and O) in A549 WT and RPAP2-KO cells (J-L) or WT and HMGB2-KO cells (M-O) following infection with PR8 (MOI=3). Data is shown as the fold change over input (0hpi). Statistical significance was assessed at each timepoint following log transformation using multiple unpaired t tests, corrected for multiple comparisons using the FDR. For all panels: Data is shown as mean of n=3 technical repeats, representative of n=3 biological repeats. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. See also Figure S4 and S5.

Article Snippet: Primary antibodies used were rabbit IZ-vinculin (EPR8185; Abcam), rabbit IZ-PB2 (GTX125926; Genetex), rabbit IZ-HMGB2 (EPR6301; Abcam), rabbit IZ-RUVBL2 (10195-1-AP; ThermoFisher Scientific), mouse IZ-tubulin (AB7291; Abcam), mouse IZ-FLAG (F1804, Sigma).

Techniques: Activity Assay, Reporter Assay, Transfection, Luciferase, Infection, Transformation Assay

Journal: bioRxiv

Article Title: Influenza A virus polymerase co-opts distinct sets of host proteins for RNA transcription or replication

doi: 10.1101/2025.06.06.658254

Figure Lengend Snippet: HMGB2 and RUVBL2 are replication-specific cofactors and RPAP2 is a transcription-specific cofactor. A) Schematic showing vRNA, cRNA, and mRNA synthesis in a minigenome setup with a WT, replication-deficient (PA E410A), or transcription-deficient (PA D108A) polymerase. B and C) cRNA:vRNA ratio (B) and mRNA:vRNA ratio (C) produced by a PR8 WT vRNP following transient knockdown of both ANP32A and ANP32B (siANP32.1 and siANP32.2), HMGB2, RUVBL2, or RPAP2. Activity of a replication-deficient vRNP (green striped) and transcription-deficient vRNP (blue striped) in cells transfected with a non-targeting siRNA (siNT) is also shown. cRNA:vRNA ratios were normalized to the ratio produced by the transcription-deficient vRNP in siNT-treated cells (B). mRNA:vRNA ratios were normalized to the ratio produced by the replication-deficient vRNP in siNT-treated cells (C). vRNA template provided was a pPolI-Firefly luciferase plasmid. Statistical significance was assessed compared to replication-deficient/transcription-deficient polymerases using an unpaired t test. D) Schematic showing vRNA, cRNA, and mRNA synthesis under conditions of a cRNP stabilization assay with cycloheximide (CHX) treatment. E-H) cRNP stabilization assay with CHX in A549 WT and HMGB2-KO cells (E and F) or WT and RPAP2-KO cells (G and H). Data is shown as fold change in segment 4 (Haemagglutinin (HA)) cRNA:vRNA ratio (E and G) and mRNA:vRNA ratio (F and H) over input (0hpi). Statistical significance was determined at each timepoint following log transformation using multiple unpaired t tests, corrected for multiple comparisons using the FDR. For (B), (C), and (E-H): Data is shown as mean of n=3 technical repeats, representative of n=3 biological repeats. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. See also Figure S6.

Article Snippet: Primary antibodies used were rabbit IZ-vinculin (EPR8185; Abcam), rabbit IZ-PB2 (GTX125926; Genetex), rabbit IZ-HMGB2 (EPR6301; Abcam), rabbit IZ-RUVBL2 (10195-1-AP; ThermoFisher Scientific), mouse IZ-tubulin (AB7291; Abcam), mouse IZ-FLAG (F1804, Sigma).

Techniques: Produced, Knockdown, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Transformation Assay