Wigington Callie P, Morris Kevin J, Newman Laura E, Corbett Anita H
From the Department of Biochemistry and.
the Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, Georgia 30322.
J Biol Chem. 2016 Oct 21;291(43):22442-22459. doi: 10.1074/jbc.M116.754069. Epub 2016 Aug 25.
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 F 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.
聚腺苷酸RNA结合蛋白（Pabs）调节基因表达的多个步骤。这个蛋白家族包括研究充分的Pabs、PABPN1和PABPC1，以及新鉴定的Pab——含锌指CCCH型结构域蛋白14（ZC3H14）。ZC3H14的突变与一种智力残疾形式有关。为了探究ZC3H14的功能，我们对缺失ZC3H14或对照Pab即PABPN1的细胞进行了全转录组分析。PABPN1的缺失影响了约17%的表达转录本，而ZC3H14仅影响了约1%的表达转录本。为了评估ZC3H14在调节靶mRNA中的功能，我们选择了编码ATP合酶F亚基C（ATP5G1）转录本的基因。敲低ZC3H14显著降低了ATP5G1的稳态mRNA水平。与ZC3H14缺失时ATP5G1周转增加的结果一致，同时敲低ZC3H14和无义介导的衰变因子UPF1可挽救ATP5G1转录本水平。此外，分级分离显示，当ZC3H14缺失时，到达细胞质的ATP5G1前体mRNA的量增加，并且ZC3H14在细胞核中与ATP5G1前体mRNA结合。这些数据支持了ZC3H14在确保ATP5G1前体mRNA的正确核加工和保留方面的作用。与ATP5G1是ATP合酶活性的限速成分这一观察结果一致，敲低ZC3H14会降低细胞内ATP水平并导致线粒体碎片化。这些数据表明，ZC3H14调节特定mRNA转录本的前体mRNA加工，并在调节细胞能量水平中起关键作用，这些观察结果对神经元的正常功能具有广泛影响。
