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.
Highlights
Cessing and is critical to ensure proper cell function [2, 3]
An RNA-binding protein that was recently linked to tissuespecific human disease is the zinc finger polyadenosine RNAbinding protein zinc finger CCCH protein 14 or ZC3H14 [12, 13], which is termed MSUT2 [14]
Patients homozygous for loss-of-function mutations in the ZC3H14 gene have a severe form of autosomal recessive intellectual disability [12], suggesting a critical role for ZC3H14 in mRNA-processing events that are necessary for proper brain function [11]
Summary
Poly(A)-binding protein; NMD, nonsensemediated decay; cNLS, classical nuclear localization signal; qRT, quantitative RT; FWD, forward; REV, reverse; IP, immunoprecipitation; PARP, poly(ADP-ribose) polymerase; ActD, actinomycin-D; OXPHOS, oxidative phosphorylation. Depletion of ZC3H14 by siRNA-mediated knockdown causes a decrease in cellular ATP levels and a severe mitochondrial fragmentation phenotype comparable with what is observed upon direct knockdown of ATP5G1 Together, these results support a role for ZC3H14 in modulating specific target mRNAs, including those important for proper mitochondrial function that could be critical in the brain
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