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. 2016 Oct 21;291(43):22442-22459.
doi: 10.1074/jbc.M116.754069. Epub 2016 Aug 25.

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA

Callie P Wigington  1   2 , Kevin J Morris  1   2 , Laura E Newman  1   2 , Anita H Corbett  3   2
Affiliations

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA

Callie P Wigington et al. J Biol Chem. .

Abstract

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function.

Keywords: ATP synthase; MSUT2; Nab2; RNA; RNA processing; RNA splicing; RNA-binding protein; ZC3H14; post-transcriptional regulation.

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Figures

FIGURE 1.
FIGURE 1.
Knockdown of ZC3H14 decreases ATP5G1 mRNA levels in all cell types examined. A, ZC3H14 is alternatively spliced to form at least four distinct protein isoforms (Iso1–4), three longer isoforms (Iso1–3), and a shorter isoform (Iso4). All isoforms contain the C-terminal (CCCH)5 zinc finger domain (blue) that confers RNA binding. Isoforms 1–3 differ from one another only in selective inclusion of exons 10–12 (teal and dark gray). These isoforms all contain an N-terminal proline tryptophan isoleucine-like (PWI-like) fold as well as a predicted cNLS. Consistent with the presence of a cNLS, Iso1–3 are all localized to the nucleus at steady state. ZC3H14 isoform 4 contains a distinct N-terminal exon (white). As the cNLS is absent from this isoform, Iso4 localizes to the cytoplasm at steady state. The ZC3H14 antibody used in this study recognizes the N-terminal domain of isoforms 1–3 (Antibody Epitope). The siRNAs employed in this study target two independent sequences within the region that encodes for the CCCH zinc fingers. B, to assess knockdown, MCF-7 cells transfected with Scramble (Scr.), ZC3H14 (ZC3), or PABPN1 (PAB) siRNA were subjected to immunoblot analysis with ZC3H14 or PABPN1 antibody and control antibodies to detect tubulin and heat shock protein 90 (HSP90). Robust knockdown of ZC3H14 (∼75–80%) and PABPN1 (∼60–75%) was detected with no effect on tubulin or HSP90 (controls). C, total RNA isolated from MCF-7 cells transfected as in B was used for cDNA generation and hybridization to the Illumina BeadChip microarray platform. A schematic is shown indicating the relative number of transcripts that show a change (>1.5-fold) in steady-state level for each knockdown with size of circle representing fraction of transcripts impacted. Significance analysis of microarrays analysis revealed that 171 out of 13,918 (∼1%) of expressed transcripts in the transfected cells were affected (increased or decreased) by knockdown of ZC3H14 (101 increased and 70 decreased), whereas PABPN1 knockdown modulated 2,375 out of 13,722 (∼17%) expressed transcripts (1,285 increased and 1,090 decreased). D, fold-change values of select affected transcripts identified by the microarray analysis were plotted against fold-changes of the same select transcripts obtained by qRT-PCR analyses. Linear regression was used to determine the R2 value of 0.91, which represents a significant correlation between the results of both analyses and validates the effect on the transcripts analyzed. E and F, total RNA isolated from MCF-7 cells treated with mock transfection (Mock), Scramble siRNA (siScr.), ZC3H14 (siZC3H14, E) or PABPN1 (siPABPN1, F) siRNA was used for cDNA generation and qRT-PCR analysis with transcript-specific primers to detect ATP5G1 and the control RPLP0 mRNA. Knockdown of ZC3H14 (E), but not PABPN1 (F), results in a significant decrease in ATP5G1 steady-state mRNA levels. G, HeLa, HEK293, MB-231, and D556 cells (left to right) were transfected with Scramble or ZC3H14 siRNA. Transfected cells were subjected to immunoblot analysis to confirm knockdown (top) with ZC3H14 and tubulin (control) antibodies as well as qRT-PCR analysis (bottom) with ATP5G1 and RPLP0 (control) primers. Robust knockdown of ZC3H14 in each cell type resulted in a significant decrease in ATP5G1 steady-state mRNA levels. Values represent the mean ± S.E. for n = 3 independent experiments. ** and *** represent p ≤ 0.01 and p ≤ 0.001, respectively.
FIGURE 2.
FIGURE 2.
Re-expression of ZC3H14 isoform 1 restores ATP5G1 transcript levels. To rescue the effect of ZC3H14 knockdown on ATP5G1 mRNA levels, MCF-7 cells were transfected with either Scramble (siScr.) or ZC3H14 (siZC3) siRNA alone or co-transfected with ZC3H14 siRNA and pcDNA3 (Vector), Myc-tagged ZC3H14 isoform 1 (Iso1), or Myc-tagged ZC3H14 isoform 4 (Iso4) for 48 h. The Myc-tagged ZC3H14 constructs harbor silent mutations in the ZC3H14 siRNA-targeting regions and are therefore refractory to siRNA knockdown. Transfected cells were subjected to immunoblot analysis (A) with ZC3H14, tubulin (control), or Myc antibody and qRT-PCR analysis (B) with primers specific to ATP5G1 and control 18s rRNA. Scramble control values are set to 1.0, and fold-reduction of ATP5G1 mRNA is represented on a log2 axis. A significant rescue of ATP5G1 mRNA upon re-expression of Myc-Iso1 but not -Iso4 is indicated by *, which represents p ≤ 0.05.Values represent the mean ± S.E. for n = 3.
FIGURE 3.
FIGURE 3.
ZC3H14 specifically regulates the ATP5G1 transcript. A, ATP5G1, ATP5G2, and ATP5G3 transcripts are transcribed from three separate genomic loci on chromosomes (Chr.) 17, 12, and 2, respectively. The ATP5G1, -2, and -3 genes and their corresponding mRNAs have varying lengths (reported in base pairs (bp) or nucleotides (nt); thin bars = introns, thicker bands = UTRs, and boxes = coding regions) and encode distinct protein products (P1, P2, and P3). ATP5G2 is alternatively spliced to form two distinct mRNAs and subsequent protein products, P2a and P2b. The encoded protein products contain identical C termini with variable N-terminal mitochondrial targeting peptides (red, with the number of amino acids indicated) that are cleaved (red lightning bolt) upon import into the mitochondria. The resulting mature protein products (dark gray) are completely identical in amino acid sequence (76 amino acids). B, total RNA isolated from MCF-7 cells was used for qRT-PCR analysis with primers specific to each of the ATP5G mRNAs. The relative value of each ATP5G mRNA was calculated by 2∧−Ct and is reported as relative units. C, MCF-7 cells transfected with Scramble control or ZC3H14 siRNA were subjected to RNA isolation and qRT-PCR analysis with primers specific to all three ATP5G mRNAs as well as the control transcript, RPLP0. Values are set to 1.0 for siScramble and normalized to RPLP0. Knockdown of ZC3H14 results in a specific and robust decrease in ATP5G1 steady-state mRNA levels. D, endogenous nuclear isoforms of ZC3H14 were immunoprecipitated from MCF-7 cells using either ZC3H14 antibody-bound protein A/G beads or control rabbit pre-immune serum-coated beads. Proteins from the input (I), unbound (UB), and bound (B) fractions were resolved on an SDS-polyacrylamide gel and subjected to immunoblotting with ZC3H14 antibody. The nuclear ZC3H14 isoforms were detected in the ZC3H14-bound fraction but not the pre-immune bound fraction. E, RNA isolated from the ZC3H14 RNA-IP was subjected to qRT-PCR analyses with GAPDH, RPLP0, ATP5G1, ATP5G2, ATP5G3, and ZC3H14 primers. mRNA levels in the ZC3H14 bound fractions were normalized to input levels and then compared by fold-enrichment over pre-immune control. Significant enrichment of ATP5G1, ATP5G3, and ZC3H14 transcripts was observed with ZC3H14 IP. Values represent the mean ± S.E. for n = 3 independent experiments. **, ***, and **** represent p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001, respectively.
FIGURE 4.
FIGURE 4.
ZC3H14 modulates the decay of ATP5G1 mRNA. A, total RNA isolated from MCF-7 cells treated with mock transfection, Scramble, or ZC3H14 siRNA was used for qRT-PCR analysis with primers that amplify ATP5G1, -2, -3, and RPLP0 pre-mRNAs as well as mature RPLP0 mRNA. Pre-mRNA levels of each transcript are normalized to levels of mature RPLP0 (set to 1.0), and no significant difference was observed in pre-mRNA levels of these transcripts upon ZC3H14 knockdown. B, MCF-7 cells were treated with the transcriptional inhibitor, ActD, and collected at the indicated time points after drug addition. Total RNA isolated from ActD-treated cells was subjected to qRT-PCR analysis with ATP5G1, ATP5G2, ATP5G3, and 18S rRNA (control) primers. mRNA levels were normalized to time 0 and are represented as % of amount present at time 0. The predicted half-lives of ATP5G1 and ATP5G2 were calculated to be ∼21.0 h, although the predicted half-life ATP5G3 was shorter at ∼13.3 h. To examine differences in stability of the ATP5G transcripts upon ZC3H14 knockdown, MCF-7 cells transfected with Scramble or ZC3H14 siRNA were treated with ActD and collected at the indicated time points after drug addition. qRT-PCR analysis of total RNA isolated from these samples with primers specific to ATP5G1, -2, -3, and RPLP0 mRNAs demonstrates a modest but significant decrease in ATP5G1 mRNA stability (C) with no difference in the decay rate of the other transcripts examined (D–F). mRNA levels were normalized to time 0 and are represented as % mRNA remaining. To determine whether ZC3H14 is involved in proper processing of ATP5G1 mRNA, MCF-7 cells were transiently transfected with Scramble, UPF1, ZC3H14, or UPF1/ZC3H14 (siBoth) siRNA. Transfected cells were subjected to immunoblot analysis (G) with ZC3H14, UPF1, and tubulin (control) antibodies and qRT-PCR analysis (H) with primers specific to ATP5G1 and control transcript, RPLP0. Double knockdown of ZC3H14 and UPF1 results in a significant rescue of ATP5G1 mRNA levels compared with ZC3H14 knockdown alone, suggesting that loss of ZC3H14 results in a pre-mRNA processing defect of ATP5G1 upstream of NMD. Data points represent the mean ± S.E. for n = 3 independent experiments. *, **, and # represent p ≤ 0.05, p ≤ 0.01, and p = 0.056, respectively.
FIGURE 5.
FIGURE 5.
ZC3H14 binds to ATP5G1 mRNA in the nucleus. To determine whether ZC3H14 knockdown impacts ATP5G1 mRNA levels in a specific compartment, MCF-7 cells were transiently transfected with either Scramble (Scr.) or ZC3H14 (ZC) siRNA and then fractionated to analyze the nuclear and cytoplasmic compartments. A, protein from whole cell (W.C.), nuclear (Nuc.), and cytoplasmic (Cyto.) samples were subjected to immunoblot analysis with ZC3H14, tubulin (cytoplasmic), and HuR (nuclear) antibodies. As expected and consistent with efficient fractionation, HuR and tubulin display primarily nuclear and cytoplasmic localizations, respectively. Consistent with a previous study demonstrating steady-state nuclear localization of ZC3H14 in HeLa cells (13), we detect ZC3H14 primarily in the nucleus of MCF-7 cells. Robust knockdown of ZC3H14 in the whole cell and nuclear fractions is shown in the lower exposure blot (Low Exp.). A higher exposure (High Exp.) of the same blot demonstrates robust knockdown of the small cytoplasmic pool of ZC3H14 in the cytoplasmic fraction as well. B, total RNA isolated from samples in A was used for cDNA generation and qRT-PCR analysis with GAPDH, ZC3H14, and ATP5G1 primers. Knockdown of ZC3H14 resulted in a robust decrease of ZC3H14 and ATP5G1 steady-state mRNA levels detected in both the nucleus and cytoplasm. To determine whether ZC3H14 interacts with ATP5G1 mRNA in the nucleus and/or cytoplasm, MCF-7 cells were subjected to nucleocytoplasmic fractionation followed by RNA-IP, as described under “Experimental Procedures.” C, proteins from the input (I), unbound (UB), and bound (B) fractions were subjected to immunoblot analysis with ZC3H14 antibody as well as HuR and tubulin antibodies to confirm efficient fractionation. We achieve robust enrichment of ZC3H14 in each compartment. As expected, HuR and tubulin are present primarily in the nuclear and cytoplasmic fractions, respectively. D, total RNA isolated from the ZC3H14 RNA-IP in each compartment was subjected to qRT-PCR analysis with GAPDH, ATP5G1, and ZC3H14 primers. mRNA levels in the ZC3H14-bound fraction of each compartment were normalized to input levels and then compared by fold-enrichment over pre-immune control. Significant enrichment of GAPDH, ATP5G1, and ZC3H14 mRNAs was observed in the nucleus; however, ZC3H14 was the only transcript significantly enriched in the cytoplasmic samples. Data points represent the mean ± S.E. for n = 3 independent experiments. *, ***, and **** represent p ≤ 0.05, p ≤ 0.001, and p ≤ 0.0001, respectively.
FIGURE 6.
FIGURE 6.
ZC3H14 interacts with ATP5G1 pre-mRNA. A, to determine whether ZC3H14 interacts with ATP5G1 pre-mRNA, RNA isolated from ZC3H14-precipitated MCF-7 cell lysates (see Fig. 3D) were subjected to qRT-PCR analysis with mature RPLP0 mRNA, RPLP0 pre-mRNA (Pre-RPLP0), mature ATP5G1 mRNA, ATP5G1 pre-mRNA (Pre-ATP5G1), and ZC3H14 primers. Consistent with the results from Fig. 3E, we observe significant enrichment of ATP5G1 and ZC3H14 mRNAs in the ZC3H14 bound fraction. Interestingly, we observe significantly higher enrichment of RPLP0 and ATP5G1 pre-mRNA levels compared with their respective mature transcripts. B, total RNA isolated from cells transfected with Scramble and ZC3H14 siRNA followed by nucleocytoplasmic fractionation (as described under “Experimental Procedures”) were subjected to qRT-PCR analysis with primers to detect ATP5G1 (left) and RPLP0 (right) pre-mRNA. A schematic of the coding exons (colored boxes) and introns (black line) of the ATP5G1 and RPLP0 transcripts are represented above the respective graph. Primers used to detect pre-mRNA and mature mRNA are represented with gray and black arrows, respectively. The nuclear/cytoplasmic (N/C) ratio of ATP5G1 pre-mRNA is significantly decreased upon ZC3H14 knockdown, likely due to the increased cytoplasmic levels of ATP5G1 pre-mRNA. The nuclear/cytoplasmic ratio of RPLP0 pre-mRNA is unchanged upon ZC3H14 knockdown. Data points represent the mean ± S.E. for n = 3 independent experiments. *, **, ***, and **** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, and p ≤ 0.0001, respectively.
FIGURE 7.
FIGURE 7.
Knockdown of ZC3H14 results in fragmented mitochondria. A, to assess cellular ATP levels, cells treated with vehicle control, the electron transport chain inhibitor, rotenone, Scramble siRNA, or siRNA targeting ATP5G1 or ZC3H14 were subjected to boiling water extraction and ATP level quantification using a luciferase-based assay. Cellular ATP levels are normalized to vehicle control or siScramble, which are both set to 1.0 and plotted as relative ATP levels. ZC3H14 knockdown results in decreased cellular ATP levels similar to that observed with rotenone treatment or knockdown of ATP5G1. B, cells treated with a mock transfection, Scramble siRNA, or ZC3H14 siRNA were harvested, and total RNA was used for qRT-PCR analyses. Primers specific to one representative nuclear-encoded mitochondrial mRNA from each OXPHOS complex as well as the control transcript, RPLP0, were used to assess any overall impact of ZC3H14 knockdown on steady-state levels of transcripts encoding OXPHOS components I, NDUFA4; II, SDHB; III, UQCRFS1; IV, CoxIV, and V, ATP5B (67). Relative mRNA values for each OXPHOS mRNA from mock transfection are set to 1.0. C, MCF-7 cells transfected with either Scramble (siScramble), ATP5G1 siRNA (siATP5G1), or ZC3H14 (siZC3H14) were fixed, permeabilized, and subjected to immunofluorescence with cytochrome c antibody. Insets are enlarged from the boxed regions of cells to better highlight mitochondrial morphology. Mitochondrial morphology in cells transfected with scrambled siRNA was indistinguishable from cells transfected with no siRNA (data not shown). D, cells from C were scored for the presence of normal or fragmented mitochondria. Data are represented as a mean averaged from three independent experiments (n = 304 for mock transfected cells, 311 for scrambled siRNA, 307 for ZC3H14 siRNA, and 307 for ATP5G1 siRNA). The difference between control cells treated with Scramble siRNA and either ZC3H14 or ATP5G1 siRNA-treated cells was statistically significant. E, MCF-7 cells treated with mock transfection, Scramble, ZC3H14, or ATP5G1 siRNA or the apoptotic inducer staurosporine (Staur.) were subjected to immunoblot analysis with PARP or tubulin antibody. The presence of cleaved PARP product only in the staurosporine-treated samples suggests that the other cell populations are not undergoing apoptosis. Values represent the mean ± S.E. for n = 3 independent experiments. ** represents p ≤ 0.01; ***, p ≤ 0.01. Images are representative of n = 3 independent experiments with at least 100 cells per experiment in each treatment group.
FIGURE 8.
FIGURE 8.
Model. Left, in cells with normal levels of ZC3H14 (Z; pink, five-fingered shape), ZC3H14 interacts with poly(A) tails throughout nuclear processing events to ensure the coordination of proper pre-mRNA (represented with gray exons and including introns) processing events and to couple these events to export, resulting in the selective export of export-competent mRNPs (represented with green, spliced exons). ZC3H14 is likely removed during the process of export (black dotted arrow). The proper production and export of ATP5G1 mRNA maintains a healthy pool of mitochondria (green ovals at bottom of image). Right, in cells with reduced ZC3H14 levels, post-transcriptional processing events are not properly coordinated, resulting in a decrease in the production of mature mRNA and an increase in improperly and/or incompletely processed pre-mRNAs in the cytoplasm and a disruption in normal mitochondrial morphology (red shapes at bottom of image).
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References

    1. Phillips T. (2008) Regulation of transcription and gene expression in eukaryotes. Nat. Education 1, 199
    1. Glisovic T., Bachorik J. L., Yong J., and Dreyfuss G. (2008) RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett. 582, 1977–1986 - PMC - PubMed
    1. Moore M. J. (2005) From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 - PubMed
    1. Fasken M. B., and Corbett A. H. (2005) Process or perish: quality control in mRNA biogenesis. Nat. Struct. Mol. Biol. 12, 482–488 - PubMed
    1. Blackinton J. G., and Keene J. D. (2014) Post-transcriptional RNA regulons affecting cell cycle and proliferation. Semin. Cell Dev. Biol. 34, 44–54 - PMC - PubMed

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