ATAC and SAGA coactivator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct

Abstract

To understand the function of multisubunit complexes it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here we demonstrate that the core modules of ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription coactivator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histones proteins. In contrast, fully assembled ATAC complex cannot be detected in the cytoplasm of mammalian cells. However, endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related coactivators, ATAC and SAGA, assemble by using co-translational pathways, but their subcellular localization, cytoplasmic abundance and functions are distinct.

Figure 1.. Schematic illustration of ATAC, SAGA complexes and pathways of co-translational assembly of protein partners

(A) Illustration of the human ATAC complex with its four subunit HAT module highlighted. (B) Illustration of the human SAGA complex. The functional modules of SAGA, such as the HAT, the deubiquitinating (DUB), the core, the splicing (SM) and the activator-binding (AM) modules are indicated. (C and D) Insect SF9 cells were either not infected (Not-infected) or co-infected with vectors expressing the six subunits of the recombinant ATAC complex (6 sub-infected): YEATS2, ZZZ3, HA-CSRP2BP, cMyc-MBIP, Flag-WDR5, and NC2β (in C) or with YEATS2, ZZZ3, HA-CSRP2BP, cMyc-MBIP, Flag-WDR5, and GST-NC2β (in D). 48h post infection whole cell extracts were made (INPUT) and anti-YEAST2 or anti-ZZZ3 IPs were carried out. The INPUT (in C and D), IPed and peptide eluted complexes were either tested by western blot analyses with the indicated antibodies (in C), or by silver staining of the 10% SDS-PAGE gels (in D). Molecular weight markers (M) are indicated in kDa. (E) HeLa cells were transfected with either with siKAT2A KAT2B (siKAT2A/2B) or siTADA2A siRNAs or not (NT). 48h post transfection NEs were prepared and an anti-ZZZ3 IP carried out. IP-ed endogenous (Endo.) ATAC subunits were analyzed by mass spectrometry. Three technical replicates were carried out and NSAF values were calculated (see also Supplemental Table 3). NSAF values were normalized to the bait of the IP (ZZZ3). The normalized NSAF values are represented as heat maps with indicated scales. ATAC complex subunits and modules are indicated on the left.

Figure 2.. Co-translational assembly of the ATAC core module

(A-F) HeLa FRT cells expressing N-terminally GFP tagged ATAC subunits (indicated in each panel with distinct colors) were treated either with cycloheximide (CHX) or puromycin (PURO). Polysome extracts were prepared, anti-GFP-coupled RNA IP (RIP) carried out and co-IP-ed RNAs were analyzed by RT-qPCR. (A) GFP-YEAST2 RIP co-immunoprecipitated its own YEATS2 mRNA (pink bar) and endogenous ZZZ3 (blue bar), CSRP2BP (green), and NC2β (orange) mRNAs. (B) GFP-ZZZ3 RIP co-immunoprecipitated its own ZZZ3 mRNA (blue bar) and endogenous YEATS2 (pink bar), as well as CSRP2BP (green bar) mRNAs. (C) GFP-CSRP2BP RIP co-immunoprecipitated its own CSRP2BP mRNA (green bar). (D) GFP-NC2β RIP co-immunoprecipitated its own NC2β mRNA (orange bar). (E) GFP-WDR5 RIP co-immunoprecipitated its own WDR5 mRNA (turquoise bar) and endogenous MBIP mRNA (magenta bar). (F) GFP-MBIP RIP co-immunoprecipitated its own MBIP mRNA (magenta bar). In all panels results of CHX treated cells are shown with black dots and results of PURO treated cells with gray triangles. Rabbit IgG was used for mock RIPs. mRNA fold enrichment is expressed as a fold change with respect to the mock RIP by using the formula ΔΔCp ^{[antl-GFP RIP/mock RIP]} Error bars ± SD are from three biological replicates (n=3). Each black dot or gray triangle represents three technical replicates. The unrelated PPIB mRNA was used as a negative control in all RT-qPCR experiments. (G) Drawings representing the results obtained in (A-F). (H) Proposed ATAC core assembly model.

Figure 3.. Co-localization of endogenous ATAC subunits with mRNAs coding for their corresponding interacting partner, and mRNAs coding for simultaneous co-TA partners

Confocal microscopy imaging was used to detect endogenous ATAC subunits with mRNAs of their interacting partners by combining single-molecule RNA FISH (smiFISH) and immunofluorescence (IF). Representative multicolor confocal images for IF-coupled smiFISH images of fixed HeLa cells. Each image is a single multichannel confocal optical slice. Co-localized spots are indicated with white rectangle and as zoom-in regions shown under every image. Scale bar (3 μm). (A-C) ZZZ3, NC2β or CTTNB1 smiFISH mRNA signal is shown in magenta; IF signal for YEATS2 protein is in green. (E-F) MBIP or CTTNB1 smiFISH mRNA signal is shown in magenta; IF signal for WDR5 protein is shown in green. (H-J) Dual color smiFISH images in HeLa cells. Cy3-smiFISH signal for ZZZ3, NC2β and CTTNB1 mRNAs is shown in magenta; ATTO488-smiFISH signal for YEATS2 mRNA is shown in green. (D, G) Boxplots showing the percentage of cytoplasmic RNA spots co-localized with protein spots in IF-smiFISH experiments. (K) Boxplots showing the percentage of cytoplasmic YEATS2 RNA spots co-localized with the indicated RNA target spots in dual-color smiFISH experiments. Each black circle represents one biological replicate. n=3 for the cells treated with cycloheximide (CHX) and n=2 for the cells treated with puromycin (PURO). For each condition, the number of cells analyzed is indicated in bracket above each boxplot. Unpaired two tailed t-tests were performed for statistical analyses between two different experimental condition (CHX and PURO). *p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001.

Figure 4.. Co-translational assembly of SAGA core subunits and co-localization of endogenous TAF12 protein with TADA1 mRNA in cytoplasm

(A- C) HeLa FRT cells expressing N-terminally GFP tagged SAGA subunits (indicated in each panel with distinct colors) were treated either with cycloheximide (CHX) or puromycin (PURO). Polysome extracts were prepared, anti-GFP-coupled RNA IP (RIP) carried out and co-IP-ed RNAs were analyzed by RT-qPCR. (A) GFP-TAF9 RIP co-immunoprecipitated its own TAF9 mRNA (yellow bar) and endogenous TAF6 (black bar), as well as TAF6L (pink bar) mRNAs. (B) GFP-TAF12 RIP co-immunoprecipitated its own TAF12 mRNA (blue bar) and endogenous TAF4 (black bar), as well as TADA1 (dark red bar) mRNAs. (C) GFP-TAF5L RIP co-immunoprecipitated its own TAF5L mRNA (green bar) and endogenous SUPT20H mRNA (orange bar). Results of CHX treated cells are represented with black dots and results of PURO treated cells were represented by gray triangles. Rabbit IgG was used as mock IP for RIP. mRNA fold enrichment is expressed as a fold change with respected to the mock RIP by using the formula ΔΔCp ^{[anti-GFP RIP/mock RIP]}. Error bars ± are from three biological replicates (n=3). Each black dot or gray triangle represents three technical replicates. The unrelated PPIB mRNA was used as a negative control in the RT-qPCR experiments. (D) Drawings representing the results obtained in (A-C). (E-G) Confocal microscopy imaging was used to detect endogenous TAF12 protein with its interacting partner mRNAs, TADA1 or TAF4, by smiFISH and IF. smiFISH signal for TADA1, TAF4 and CTTNB1 mRNAs is shown in magenta; IF signal for TAF12 protein in shown green. Each image is a single multichannel confocal optical slice. Co-localized spots are indicated with white rectangle and zoom-in regions are shown under every panel. Scale bar (3 μm). (H) Boxplot showing the percentage of cytoplasmic RNA spots co-localized with protein spots in IF-smiFISH experiments. Each black circle represents one biological replicate. n=3 for the cells treated with cycloheximide (CHX) and n=2 for the cells treated with puromycin (PURO). For each condition, the number of cells analyzed is indicated in bracket above each boxplot. Unpaired two tailed t-test was performed for statistical analyses between two different experimental condition (CHX and PURO). *p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001.

Figure 5.. Human SAGA complex can be isolated from both nuclear and cytoplasmic compartments, while human ATAC is only detectable in the nucleus

(A) Mass spectrometry analyses of KAT2A and TADA3 IPs carried out using either nuclear (NE) or cytoplasmic (CE) extracts (as indicated) prepared from human HeLa cells. NSAF values were calculated and normalized to SGF29 values. (B) Mass spectrometry analyses of TADA2A, YEATS2 and ZZZ3 IPs carried out using NE and CE (as indicated) prepared from human HeLa cells (left panel), TADA2A and ZZZ3 IPs carried out using NE and CE prepared from either human HEK293T (middle panel), or mES (right panel) cells. NSAF values were calculated and normalized to MBIP values in all NE IPs. (C) Mass spectrometry analysis of TADA2B, SUPT20H and ATXN7L3 IPs carried out using NE and CE (as indicated) prepared from either HeLa cells (left panel), TADA2B and SUPT20H IPs carried out using NE and CE prepared from either HEK293T (middle panel) or mES (right panel) cells. NSAF values were calculated and normalized to TAF6L values in all IPs. In panels (A-C) three technical replicates were carried out per IP (see also Supplemental Table 4). NSAF values were calculated and normalized to SGF29 (in A), MBIP (in B) and TAF6L (in C). Normalized NSAF results are represented as heat maps with indicated scales. Dotted boxes indicate the bait protein for a given IP. The known modules of the SAGA complex, such as HAT, DUB, core, structural, splicing (SM) and the activator-binding (AM) modules are indicated. ATAC HAT and core modules are indicated. In (A) the shared HAT subunits are highlighted and the specific HAT subunits indicated. Dotted lines separate functional modules of ATAC or SAGA complexes. (D) Live-cell measurement of subcellular distribution in GFP-TAF5L (SAGA subunit) and GFP-ZZZ3 (ATAC subunit) HeLa FRT cells. Cells were induced to express the respective GFP-fusion protein for 8 hours before imaging. Two representative GFP Z maximum intensity projections are shown for each cell line with overlayed nuclear outlines in green. The mean GFP cytoplasmic/nuclear intensity ratio for each cell is plotted on the right.

Figure 6.. The SAGA complex acetylates non-histones proteins in the cytoplasm

(A) HeLa cells were transfected with of siNon-targeting (siNonT), siTADA2B and siKAT2A/KAT2B (siKAT2A/2B) siRNAs for 48h, cytoplasmic extracts (CEs) prepared, and anti-SUPT3H IP-coupled mass spectrometry analysis was carried out. NSAF values were calculated (see Supplemental Table 5 for proteins found in each IP). NSAF values were normalized to the bait SUPT3H and the results are represented as heat maps with indicated scales. The different modules of the SAGA complex are indicated. (B) Acetylated-lysine IPs were carried out from siNonT, siTADA2B or siKAT2A/2B treated CEs and analyzed by mass spectrometry (see also Supplemental Table 6 for acetylated proteins found in each IP carried out from CE extracts, and their corresponding acetylated lysine residues). Left panel, MA plot represents log₂ fold-change (FC) of siTADA2B over siNonT (Y axis), versus siNonT normalized signal intensity in log₂ (X axis). Right panel, MA plot represents log2 FC of siKAT2A/2B over siNonT (Y axis), versus siNonT normalized signal intensity in log₂ (X axis). Peptides which have upregulated acetylation levels are indicated with orange dots. Peptides which have downregulated acetylation levels are indicated with blue dots. Peptides that have upregulated or downregulated acetylation levels in both KD (TADA2B and KAT2A/KAT2B) conditions are indicated with black circles in each category. (C) Venn diagram shows number of peptides that had significantly downregulated (DR) acetylation levels either in TADA2B (blue), and in KAT2A/2B (purple) KD conditions. (D) Log₂ difference of acetylation levels of α-synuclein or S100A11 in both KD condition. (E, F) In vitro acetylation (AT) assay. KAT2A wt, KAT2B wt and their corresponding catalytic dead mutants (mut) were purified from baculovirus infected Sf9 cells, and GFP-α-synuclein and GFP-S100A11 from transfected HeLa cells. (E) In vitro acetylation of GFP-α-synuclein by KAT2A and KAT2B acetyltransferases. The order of protein addition in the reaction mixtures is depicted on the top. The upper membrane shows western blot analysis (WB) using an anti-acetylated lysine antibody and the lower membrane using an anti-α-synuclein antibody. (F) In vitro acetylation of GFP-S100A11 by KAT2A and KAT2B acetyltransferases. The order of protein addition in the reaction mixtures is depicted on the top. The upper membrane was immunoblotted with anti-acetylated lysine antibody and the lower membrane with anti-S100A11 antibody. In (E-F) arrows indicate the corresponding proteins. (G) RT-qPCR analysis of mRNA levels of SNCA and S100A11 upon siTADA2B or siKAT2A/2B mediated KDs. Dots represent biological replicates, n=2. Log₂FC was calculated with 2^−ΔΔCT method. GAPDH mRNA was used as an internal control. (H) Western blot analyses of α-synuclein or S100A11 protein levels in both NEs and CEs upon control and siTADA2B or siKAT2A/2B KDs. The left membrane was incubated with anti-α-synuclein antibodies and right membrane with anti-S100A11 antibodies. Arrows indicate the corresponding proteins. Ponceau S staining was used as a loading control. In (E, F, H) molecular weight markers (Mw) are indicated in kDa.