Sasaki Kenta, Ono Mana, Takabe Kaori, Suzuki Atsushi, Kurihara Yasuyuki
Laboratory of Molecular Biology, Graduate School of Engineering, Yokohama National University, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
Laboratory of Molecular Biology, Graduate School of Engineering, Yokohama National University, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
Gene. 2018 May 30;657:1-8. doi: 10.1016/j.gene.2018.03.005. Epub 2018 Mar 2.
DAZAP1 is an evolutionarily conserved RNA-binding protein expressed in many tissues in mice and humans. DAZAP1-knockout mice carrying a partial loss-of-function (hypomorphic) allele exhibited severe deficiencies in spermatogenesis and cell growth, indicating that DAZAP1 plays a pivotal role in the development of germ and somatic cells. We have identified cox6c mRNA, which encodes a subunit of complex IV of the mitochondrial respiratory chain, as a target transcript regulated by DAZAP1. We found that DAZAP1 bound to cox6c mRNA derived from either the genomic DNA or a genome-type expression vector in cells, but not to cox6c mRNA derived from an intronless expression vector. Interestingly, the presence of the last intron was sufficient for DAZAP1 binding to the mRNA, suggesting specific intron dependent DAZAP1 loading onto cox6c mRNA. Overexpression of DAZAP1 resulted in the accumulation of cox6c pre-mRNA for all introns, implying that DAZAP1 reduces pre-mRNA splicing efficiency. In addition, the reduction of mature cox6c mRNA levels led to decreases in the COX6C protein levels. Both DAZAP1 knockdown and COX6C overexpression retarded cell growth. The lines of evidence presented here reveal that DAZAP1 is a negative regulator of pre-mRNA splicing and may control energy production in mitochondria by regulating COX6C expression. The DAZAP1 functions described in this study may also account for the phenotypes observed in the DAZAP1 hypomorphic mice.
DAZAP1是一种在进化上保守的RNA结合蛋白，在小鼠和人类的许多组织中都有表达。携带部分功能丧失（亚效等位基因）的DAZAP1基因敲除小鼠在精子发生和细胞生长方面表现出严重缺陷，这表明DAZAP1在生殖细胞和体细胞的发育中起关键作用。我们已确定编码线粒体呼吸链复合体IV一个亚基的cox6c mRNA是受DAZAP1调控的靶转录本。我们发现DAZAP1能与细胞中来源于基因组DNA或基因组型表达载体的cox6c mRNA结合，但不能与来源于无内含子表达载体的cox6c mRNA结合。有趣的是，最后一个内含子的存在足以使DAZAP1与mRNA结合，这表明DAZAP1特异性地依赖内含子加载到cox6c mRNA上。DAZAP1的过表达导致所有内含子的cox6c前体mRNA积累，这意味着DAZAP1降低了前体mRNA的剪接效率。此外，成熟的cox6c mRNA水平降低导致COX6C蛋白水平下降。DAZAP1的敲低和COX6C的过表达均会阻碍细胞生长。本文提供的一系列证据表明，DAZAP1是前体mRNA剪接的负调控因子，可能通过调节COX6C的表达来控制线粒体中的能量产生。本研究中描述的DAZAP1功能也可能解释了在DAZAP1亚效小鼠中观察到的表型。
