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. 2018 Jan 2;22(1):44-58.
doi: 10.1016/j.celrep.2017.12.037.

Characterizing ZC3H18, a Multi-domain Protein at the Interface of RNA Production and Destruction Decisions

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Free PMC article

Characterizing ZC3H18, a Multi-domain Protein at the Interface of RNA Production and Destruction Decisions

Kinga Winczura et al. Cell Rep. .
Free PMC article

Abstract

Nuclear RNA metabolism is influenced by protein complexes connecting to both RNA-productive and -destructive pathways. The ZC3H18 protein binds the cap-binding complex (CBC), universally present on capped RNAs, while also associating with the nuclear exosome targeting (NEXT) complex, linking to RNA decay. To dissect ZC3H18 function, we conducted interaction screening and mutagenesis of the protein, which revealed a phosphorylation-dependent isoform. Surprisingly, the modified region of ZC3H18 associates with core histone proteins. Further examination of ZC3H18 function, by genome-wide analyses, demonstrated its impact on transcription of a subset of protein-coding genes. This activity requires the CBC-interacting domain of the protein, with some genes being also dependent on the NEXT- and/or histone-interacting domains. Our data shed light on the domain requirements of a protein positioned centrally in nuclear RNA metabolism, and they suggest that post-translational modification may modulate its function.

Keywords: CBC; NEXT; RNA decay; RNA exosome; ZC3H18; histones; transcription.

Figures

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Figure 1
Figure 1
Interaction Profiling of ZC3H18 Identifies Histones (A) SDS-PAGE gel of proteins co-precipitating with ZC3H18-3xF at the indicated buffer compositions. Two major isoforms of the ZC3H18-3xF bait protein are indicated with red and green dots for the fast- and slow-migrating forms, respectively. Denoted with color code are also CBCN complex components MTR4, ARS2, ZCCHC8, and CBP80 as well as histones. The identity of the indicated bands was established by MS analysis. (B and C) Scatterplots presenting MS analysis of co-purified CBCN components (B) and core histones (C) in the ZC3H18-3xF AC experiments performed in 100, 300, and 500 mM NaCl-containing buffers (indicated by red, blue, and green dots, respectively). Exact buffer compositions are shown below the plots. The y axes display protein abundances estimated as the ratio between a protein’s mean peptide intensity from two biological experiments and its molecular weight and normalized to the abundance of ZC3H18-3xF bait protein. The x axes display RNase A/T1 resistance calculated as the ratio between protein abundances in RNase A/T1-treated versus untreated samples.
Figure 2
Figure 2
ZC3H18 Binding to Histones Is Specific and Phosphorylation Dependent (A) SDS-PAGE gel of proteins co-precipitating with color-coded ZC3H18, PABPN1, NUP88, and NUP98 proteins expressed with a 3xF tag from HEK293 Flip-In T-Rex cell lines. As a control the HEK293 parental cell line was also used. AC was carried out in three different NaCl buffers (20 mM HEPES [pH 7.4], 0.5% Triton X-100 and 100, 300, or 500 mM NaCl, respectively) as indicated. 3xF-tagged proteins are indicated, based on their expected molecular weights, with color-coded dots and corresponding arrows. Only the ZC3H18-3xF construct co-purified histones in the 500 mM NaCl buffer (indicated by black vertical line in lane 3). (B) SDS-PAGE gel showing the ZC3H18 isoforms and co-precipitating proteins after a two-step ZC3H18-3xF AC in 100 and 600 mM-NaCl conditions (see main text for details). Migration of ZC3H18-3xF fast- and slow-migrating forms as well as CBCN complex components are indicated as in (A). Note that histones are bound only by the slow-migrating form of ZC3H18-3xF. (C) SDS-PAGE gel of proteins co-precipitating with ZC3H18-3xF in 100, 300, and 600 mM-NaCl containing buffers supplemented (+) or not (−) with PPI. ZC3H18-3xF isoforms denoted as in (A). (D) ChIP-PCR analysis of ZC3H18 occupancy along the MYC gene and MYC PROMPT using amplicons from top schematics. IgG ChIP was used to assess background binding. Data are displayed as mean ± SD of two biological replicates. Significance levels between the ZC3H18 and IgG samples were assessed by a two-way ANOVA test with obtained p values presented above the bars. (E) ChIP-PCR analysis of ZC3H18 occupancy at promoters of transcriptionally active (GAPDH, U12, BRCA1, PSMD3, MRPS15, PALB2, ZNHIT3, KRAS, and SNRPB2) and repressed (IFRG28, MYT1, and KCNA1) genes. IgG ChIP was used to assess background binding. Data are displayed as mean ± SD of two technical replicates of the representative experiment. Significance levels between the ZC3H18 and IgG samples were calculated using a two-way ANOVA test and are denoted with asterisks corresponding to the following p value ranges: p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001.
Figure 3
Figure 3
Domain Mapping of ZC3H18 (A) Schematic representation of the FL and Z1-Z7 mutant variant ZC3H18 constructs. Dashed line in Z7 construct indicates deletion. CC, coiled coil; ZF, zinc finger; Ser-rich, serine-rich region (UniProt: Q86VM9; www.uniprot.org). All constructs were C-terminally fused to 3xF. (B) Western blotting analysis of the indicated proteins co-purifying with ZC3H18-3xF FL and mutant variants in 100 mM-NaCl (top panel) or 600 mM-NaCl (bottom panel) AC conditions. In the low-salt condition, input and eluate samples were probed with antibodies against the CBCA components ARS2 and CBP80 and the NEXT components MTR4, ZCCHC8, and RBM7 as well as α-FLAG antibodies against the 3xF-tagged constructs. In the high-salt condition, ZC3H18-3xF variants and histones were visualized using Coomassie staining. Note that the lower band in the α-MTR4 western analysis, denoted with an asterisk, corresponds to ARS2 signal from a previous exposure of the same membrane. (C) Schematic representation of interaction domains on ZC3H18.
Figure 4
Figure 4
ZC3H18 Depletion Affects Transcription of a Subset of Protein-Coding Genes (A) RNA-seq-derived differential expression changes of exonic reads between ZC3H18 depletion and control (EGFP) libraries. The y axis displays the log2 fold change of read counts, and the x axis displays the log10-transformed exonic read count per gene. Data were computed by the DESeq2 software (Love et al., 2014). All individual genes are shown in gray and significantly called transcripts (padj = FDR < 0.1) are colored red. Of the n = 18,731 genes shown, n = 1,756 and n = 2,110 were significantly up- and downregulated, respectively. BRCA1, BRCA2, and OMA1 RNAs are highlighted. (B) As in (A) but based on reads from intronic regions of each gene, scaled using sizing factors from exonic reads. Of the n = 17,872 genes shown, n = 1,277 and n = 905 were significantly up- and downregulated, respectively. (C) Scatterplot showing RNA-seq log2 fold changes of exonic (from A) versus intronic (from B) regions for individual genes (n = 16,157, Pearson correlation coefficient r2 = 0.56). RNAs that were significantly up- or downregulated (padj = FDR < 0.1) at exon or intronic levels are colored yellow. RNAs that were significantly affected in both exon and intron read counts are colored red. BRCA1, BRCA2, and OMA1 RNAs are highlighted. (D) Scatterplot showing Pol II ChIP-seq log2 signals in ZC3H18 depletion (y axis) relative to control (siFFL) (x axis) samples. Genes expressing RNAs that were significantly (padj = FDR < 0.1) up- or downregulated in the RNA-seq intronic analysis are colored blue and red, respectively. BRCA1, BRCA2, and OMA1 genes are highlighted. (E) Heatmap of hierarchical clustering analysis of log2 fold changes of RNA-seq exonic (left part) or intronic (right part) reads from ZC3H18, CBP80, ARS2, RBM7, RRP40, and ZCCHC8 depletion relative to control samples (denoted by siXXXX below the figure). RNAs with padj < 0.01 and log2 fold change >1 or log2 fold change <−1 were included (n = 2,626).
Figure 5
Figure 5
Functional Impact of ZC3H18 Protein Interaction Domains (A) Schematic representation of rescue experiments performed in HEK293 cell lines containing stably integrated and inducible ZC3H18-3xF FL and mutant variants. Endogenous ZC3H18 depletion was subjected to possible suppression by exogenous expression of ZC3H18-3xF variants. (B) qRT-PCR analysis of indicated mRNA levels in total RNA samples from HEK293 cells depleted for endogenous ZC3H18 and expressing the indicated exogenous and ZC3H18-3′ UTR siRNA-resistant variants. Data are displayed as mean ± SD of at least three biological replicates normalized to the control (EGFP) sample and GAPDH mRNA. Significance levels between the mean values of mRNA levels in ZC3H18 depletion versus rescued samples were calculated using a two-way ANOVA test and are denoted with asterisks corresponding to the following p value ranges: p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. (C) qRT-PCR analysis as in (B) but using primers detecting U11 and U12 snRNA 3′ extended regions.
Figure 6
Figure 6
Dual Roles of ZC3H18 in Nuclear RNA Metabolism Schematic representation of the dual engagement of ZC3H18 in RNA transcription and decay processes. Left: the C terminus of ZC3H18 interacts with the CBCA complex to regulate protein-coding gene transcription directly or indirectly. Right: short non-coding RNAs, such as 3′ extended snRNAs, are targeted ZC3H18 dependently in a process requiring both CBCA and NEXT interaction of ZC3H18. Such RNA decay can also occur ZC3H18 independently via direct NEXT exosome targeting. See the main text for details. C, CBCA-interacting domain; H, histone-interacting domain; N, NEXT-interacting domain. Question mark indicates the elusive role of the histone-interacting domain and its modification in modifying the functionality of ZC3H18.

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