Functional analysis of a novel GTP-binding protein involved in apoptosis.

我们克隆了ERA的人类同源物（H-ERA）和小鼠同源物（M-ERA）的整个编码区域的cdna。ERA的哺乳动物同源结构包括一个典型的GTPase/ gtp结合结构域和一个被认为是RNA结合结构域的K同源结构域（KH）。我们用野生型H-ERA或各种H-ERA突变体进行转染实验。具有GTPase结构域氨基酸取代突变的H-ERA诱导HeLa细胞凋亡，并被Bcl-2的表达阻断。缺失含有部分KH结构域的c端可减轻H-ERA突变体的凋亡，提示该结构域在H-ERA功能中的重要性。我们还通过将RNA均聚物固定在微球或重组H-ERA蛋白上的下拉实验证明了H-ERA的RNA结合活性。因此，H-ERA可能通过其GTPase/GTP结合域在调控凋亡信号传导中发挥重要作用。为了阐明真核ERA的生理功能，我们对鸡DT40细胞中的ERA （GdERA）进行了遗传分析。GdERA的消耗降低了细胞的生长速度，并伴有凋亡细胞的积累。细胞周期分析表明，GdERA的消除在G1期引起阻滞，而不是在M期，这突出了与原核ERA相比，脊椎动物ERA在细胞周期进程中的独特作用。此外，人类ERA （HsERA）挽救了gdera缺陷细胞的表型，而剥夺RNA结合活性的HsERA突变体则没有。这些数据表明，脊椎动物的ERA通过一种未知的分子机制调节G1期的进展，该机制涉及ERA的RNA识别。

We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. Thus, H-ERA may play an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain. To elucidate the physiological function of eukaryotic ERA, we have performed a genetic analysis of chicken ERA (GdERA) in DT40 cells. Depletion of GdERA diminished the growth rate of the cells, accompanied by an accumulation of apoptotic cells. The analysis of cell cycle indicates that the elimination of GdERA caused arrest at G1 phase, but not at M phase, which highlights the distinct role of vertebrate ERA in the cell cycle progression compared to prokaryotic ERA. Furthermore, human ERA (HsERA) rescued the phenotype of GdERA-deficient cells, whereas a mutant of HsERA deprived of RNA binding activity did not. These data suggest that vertebrate ERA regulates the G1 phase progression via an as yet unknown molecular mechanism, which involves RNA recognition by ERA.