Fig 2: TAF7 binds a broad spectrum of RNA species.(A) Heatmap displaying the differential expression patterns of genes (with a fold change >2 and Padj <0.01) in FLAG RIP-seq and RNA-seq of HeLa TAF7 (WT). (B) Volcano plot of mRNA expression in FLAG RIP-Seq versus RNA-seq of HeLa TAF7 (WT) cells. Genes with a fold change > 2, and Padj < 0.01 are labeled in red. (C) Metagene analysis of PAR-CLIP T-to-C mutation site distribution across mRNA features, including 5'UTR, CDS (Coding Sequence), and 3'UTR. (D) Structure of TAR RNA with sites of mutation indicated in red. (E) TAF7-TAR interaction by EMSA. Increasing amounts of purified HIV-1 Tat proteins (50 and 100 pmol) or TAF7 proteins (5 , 10 , and 20 pmol) were incubated with WT or mutant TAR RNA (TARmut). The bands corresponding to free RNA and Tat-TAR and TAF7-TAR complexes are indicated by arrows. (F) Haspin mRNA, SMARCC2 mRNA, and DPP9 mRNA were immunoprecipitated with TAF7 in HeLa cells. The abundance of mRNA immunoprecipitated by TAF7 was measured in real-time PCR and compared with the abundance of mRNA immunoprecipitated by control mouse IgG. Results are representative of three independent experiments with a minimum of two technical replicates per experiment.

Fig 3: Depletion of cytoplasmic TAF7 leads to a reduction of protein synthesis from TAF7 RNA targets.(A) SUnSET analysis of global protein synthesis in MEF TAF7fl/fl cells depleted of endogenous TAF7 by Cre and overexpressing TAF7 (WT) or TAF7 (?NES). Following puromycin (10 µg/ml) treatment as described, incorporation was calculated from FACS analysis of puromycin-labeled cells. Data are presented as means ± SD, **P < 0.01. (B) SUnSET analysis of global protein synthesis in HeLa S3 cells depleted of endogenous TAF7 by siRNA and overexpressing TAF7 (WT) or TAF7 (?NES). Following puromycin (10 µg/ml) treatment as described, incorporation was calculated from FACS analysis of puromycin-labeled cells. Data are presented as means ± SD, *P < 0.05. (C) IB analysis of Haspin levels in MEF TAF7fl/fl TAF7 (WT) + Cre and MEF TAF7fl/fl TAF (?NES) + Cre nuclear extracts. TBP was used as a control. The Haspin/TBP values were calculated from three independent IB experiments. Error bars indicate means ± SD, **P < 0.01. (D) IB analysis of SMARCC2 levels in MEF TAF7fl/fl TAF7 (WT) + Cre and MEF TAF7fl/fl TAF7 (?NES) + Cre nuclear extracts. TBP was used as a control. The SMARCC2/TBP values were calculated from three independent IB experiments. Error bars indicate mean ± SD, **P < 0.01. (E) IB analysis of DPP9 levels in MEF TAF7fl/fl TAF7 (WT) + Cre and MEF TAF7fl/fl TAF7 (?NES) + Cre total cell extracts. Actin was used as a control. The DPP9/actin values were calculated from three independent IB experiments. Error bars indicate means ± SD, *P < 0.05.

Fig 4: Disruption of TAF7 RBD leads to a retention of TAF7 RNA targets in the nucleus and a reduction of protein synthesis from TAF7 RNA targets.(A) Mutation of TAF7 RBD impaired export of RNA targets. TAF7 (RBD/NLS) mutant and TAF7 (WT/NLS) were transduced into HeLa cells depleted of endogenous TAF7 by siRNA. Nuclear and cytoplasmic levels of Haspin, SMARCC2, and DPP9 mRNAs were measured by real-time PCR. Nuclear/cytoplasmic mRNA ratios were calculated from at least three independent experiments with a minimum of two technical replicates per experiment. Data are means ± SD, **P < 0.01, *P < 0.05. (B) Export of HIV-1 LTR-luciferase RNA is impaired in TAF7 RBD mutant. HIV-1 LTR-luciferase construct was transiently transfected into HeLa TAF7 (WT/NLS) and HeLa TAF7 (RBD/NLS) cells depleted of endogenous TAF7 by siRNA. Nuclear and cytoplasmic levels of luciferase mRNAs were measured by real-time PCR. Nuclear/cytoplasmic mRNA ratios were calculated from three independent experiments with a minimum of two technical replicates per experiment. Data are means ± SD, *P < 0.05. (C) Protein synthesis is reduced with TAF7 RBD mutant. TAF7 (RBD/NLS) mutant and TAF7 (WT/NLS) were transduced into MEF TAF7fl/fl cells depleted of endogenous TAF7 by Cre and protein synthesis measured by SUnSET. Results are shown as relative puromycin incorporation in MFI. Data are means ± SD, **P < 0.01. (D to F) IB analysis of Haspin, SMARCC2, and DPP9 levels in HeLa TAF7 (WT/NLS) + siRNA and HeLa TAF7 (RBD/NLS) + siRNA extracts. TBP was used as a control for Haspin and SMARCC2 in nuclear extracts; actin was used as a control for DPP9 in total cell extracts. Error bars indicate means ± SD from three independent experiments, **P < 0.01, *P < 0.05.