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

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 ϳ17% of expressed transcripts, whereas ZC3H14 affected only ϳ1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F 0 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.

The polyadenosine RNA-binding protein ZC3H14 interacts with the THO complex and coordinately regulates the processing of neuronal transcripts

“…Transcript levels were normalized to input and then to IgG control. Using a candidate-based approach, we tested for enrichment of RNA targets using a previously identified ZC3H14 target transcript Atp5g1 ( 9 ) and other important neuronal transcripts with potential links to ZC3H14 (unpublished results), Psd95 ( Dlg4 ) and Mapt . The Atp5g1, Psd95 and Mapt mRNA are all significantly enriched in ZC3H14, THOC5 and ALYREF precipitations as compared to the IgG control (Figure 4B – D ).…”

“…Like Nab2, ZC3H14 is primarily localized to the nucleus and both Nab2 and ZC3H14 bind with high affinity to polyadenosine RNA ( 15 , 16 ). Although Nab2 and ZC3H14 bind to polyadenosine RNA tracts ( 17 ) and therefore could bind to all mRNA transcripts, previous work has shown that loss of ZC3H14 only alters the steady-state levels of a small number of transcripts ( 9 ). This finding focuses the regulation of mRNA processing by ZC3H14 to a select group of transcripts.…”

“…As previously shown for ZC3H14, we demonstrate that THO components are required for proper control of bulk poly(A) tail length. As these factors do not affect all RNAs ( 9 , 27 , 28 ), we have identified two transcripts Atp5g1 , which encodes a component of the ATP synthase machinery ( 29 ), and Psd95 (Dlg4) , which encodes a component of the neuronal post synaptic density ( 30 ), as shared targets of ZC3H14 and the THO complex. This work reveals that ZC3H14 and the THO complex are required to coordinate nuclear processing of Atp5g1 and Psd95 mRNAs with export from the nucleus.…”

Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a

“…Nuclear samples were incubated on ice for 20 min and then centrifuged for 10 min at 15,600 × g . The supernatant (nuclear fraction) was separated from the pellet ( 34 ). After preclearance with protein G-Sepharose (GE Healthcare), the lysates were incubated with anti-Arid5a or anti-IgG at 4 °C for 8 h. The resin was washed three times with RNA immunoprecipitation buffer (50 mM Tris⋅HCl pH 7.4, 100 mM NaCl, and 0.5% Nonidet P-40 in diethylpyrocarbonate-treated water).…”

Targeting the nuclear RNA exosome: Poly(A) binding proteins enter the stage

“…59,63,75 Finally, the zinc-finger protein ZC3H14, a human homolog of Nab2p, was recently ascribed an RNA protective function, 76 and like Nab2p, ZC3H14 was suggested to control poly(A) tail length and modulate processing of pre-mRNAs. 77 An additional degradative role in RNA maturation has also recently been ascribed to PABPN1. Interestingly this occurs in a PAXT-independent manner and instead of the nuclear exosome involves the poly(A)-specific ribonuclease PARN.…”