Fig 1: TAF7 localizes in both the nucleus and the cytoplasm; cytoplasmic TAF7 is associated with RNA polysomes.(A) Immunofluorescence of TAF7 with anti-TAF7 (red) and DAPI (nuclear stain, blue) in HeLa cells. Scale bars, 10 μm. (B) Anti-TAF7 immunoblotting in HeLa nuclear and cytoplasmic fractions. Nuclear TBP and cytoplasmic β-tubulin assessed purity of respective fractions. (C) Enrichment of cytoplasmic TAF7 in HeLa cells as described in Materials and Methods. Error bars, means ± SD from three independent experiments. (D) FPLC column fractionation of HeLa cytoplasmic TAF7. HeLa TAF7 (WT) cytoplasmic extracts were subjected to Superose 6 gel-filtration chromatography; relative abundance of TAF7 and TBP in each fraction was calculated from results of immunoblotting with anti-TAF7 and anti-TBP. PC: recombinant TAF7 or TBP. (E) Proximity ligation assays (PLA) of TAF7 with RPL5 and RPL8. PLA associations, red spots; nuclear DAPI stain, blue. Scale bars, 10 μm. (F) Quantitation of PLA associations. PLA spots per cell were calculated on 400 to 500 cells. Data are means ± SD, **P < 0.01. (G) Polysome fractionation of HeLa TAF7 (WT) cytoplasmic extracts. Cytoplasmic extracts, treated with RNase inhibitor, were subjected to sucrose gradient centrifugation; fractions were analyzed by immunoblotting with anti-TAF7 and anti-RPL5 (bottom). (H) TAF7 binds RNA in vitro. Recombinant TAF7 was incubated with HeLaS3 total RNA and immunoprecipitated with anti-TAF7. BSA was the control. Precipitated RNAs were 32P 3′ end-labeled and resolved in RNA gels. (I) TAF7 binds RNA in cellulo. HeLa TAF7(WT) extracts were immunoprecipitated with anti-TAF7 or control mouse IgG. Precipitated RNA was 3′ end-labeled and resolved in RNA gels. Bottom, TAF7 recovery in the IP. (J) Truncation of TAF7 at amino acid 129 abrogates RNA binding in cellulo. Extracts from HeLaS3 cells expressing empty vector, TAF7 (WT), or TAF7 (1 to 129) were immunoprecipitated with anti-FLAG. TAF7-bound RNAs were 3′ end-labeled and examined in RNA gels. Arrow, nonspecific band. Bottom, FLAG-TAF7 recovery.
Fig 2: TAF7 contains an RBD and nuclear localization and nuclear export signals.(A) Schematic showing TAF7 domains [activation domain (AD), domains phosphorylated by TAF1/CDK9 and CDK7], RBD, nuclear localization signal (NLS), nuclear export signal (NES), and locations of deletions and mutations. Residues mutated to alanine are underlined. (B) Mapping of TAF7 RBD. TAF7 mutants were incubated in vitro with HeLa S3 total RNA and immunoprecipitated with anti-TAF7 antibody. Recovered RNAs were 3′ end-labeled with 32P and examined in an RNA gel. Bottom, TAF7 recovery. (C) Immunofluorescence with anti-HA antibody of TAF7 (WT), TAF7 (∆NES), and TAF7 (RBD) (red) and DAPI (nuclear stain, blue) in MEF cells overexpressing HA-tagged TAF7 proteins. Scale bars, 10 μm. (D) Quantitation of HA-TAF7 immunofluorescence showing enrichment of TAF7 (RBD) and TAF7 (∆NES) in the cytoplasm and nucleus, respectively. Ratios of cytoplasmic TAF7 to total TAF7 were calculated on >300 cells. Error bars, means ± SD, **P < 0.01. (E) Immunoblotting (IB) analysis of TAF7 (WT), TAF7 (∆NES), and TAF7 (RBD) in nuclear and cytoplasmic fractions from MEFs overexpressing HA-tagged proteins. (F) Determination of cytoplasmic enrichment of TAF7 (WT), TAF7 (∆NES), and TAF7 (RBD) in MEFs overexpressing HA-tagged proteins. Cytoplasmic/total ratios of TAF7 were calculated from three independent experiments. Error bars, means ± SD, **P < 0.01, *P < 0.05. (G) PLA between TAF7 and RPL5 and RPL8 in HeLa cells expressing TAF7 (WT/NLS) and TAF7 (RBD/NLS). Red spots, PLA reactions; DAPI (blue), nucleus; scale bars, 10 μm. (H) Quantitation of PLA. PLA spots per cell were calculated on 500 to 600 cells for each interaction. Data are means ± SD, **P < 0.01. (I) FLAG-IP of HeLa TAF7 (WT) cells in the presence of RNase inhibitor or RNase followed by immunoblotting. Cytoplasmic lysates were input. (J) TAF7 interacts with RPL8 in vitro. Recombinant FLAG-TAF7 was used to pull down rRPL8 protein in the presence and absence of RNA, respectively.
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