Grima Dominic P, Sullivan Melanie, Zabolotskaya Maria V, Browne Cathy, Seago Julian, Wan Kay Chong, Okada Yoshio, Newbury Sarah F
Brighton and Sussex Medical School, Medical Research Building, University of Sussex, Falmer, Brighton BN1 9PS, U.K.
Biol Cell. 2008 Dec;100(12):687-701. doi: 10.1042/BC20080049.
Ribonucleases have been well studied in yeast and bacteria, but their biological significance to developmental processes in multicellular organisms is not well understood. However, there is increasing evidence that specific timed transcript degradation is critical for regulation of many cellular processes, including translational repression, nonsense-mediated decay and RNA interference. The Drosophila gene pacman is highly homologous to the major yeast exoribonuclease XRN1 and is the only known cytoplasmic 5'-3' exoribonuclease in eukaryotes. To determine the effects of this exoribonuclease in development we have constructed a number of mutations in pacman by P-element excision and characterized the resulting phenotypes.
Mutations in pacman resulted in flies with a number of specific phenotypes, such as low viability, dull wings, crooked legs, failure of correct dorsal/thorax closure and defects in wound healing. The epithelial sheet movement involved in dorsal/thorax closure is a conserved morphogenetic process which is similar to that of hind-brain closure in vertebrates and wound healing in humans. As the JNK (c-Jun N-terminal kinase) signalling pathway is known to be involved in dorsal/thorax closure and wound healing, we tested whether pacman affects JNK signalling. Our experiments demonstrate that pacman genetically interacts with puckered, a phosphatase that negatively regulates the JNK signalling pathway.
These results reveal that the 5'-3' exoribonuclease pacman is required for a critical aspect of epithelial sheet sealing in Drosophila. Since these mutations result in specific phenotypes, our data suggest that the exoribonuclease Pacman targets a specific subset of mRNAs involved in this process. One of these targets could be a member of the JNK signalling pathway, although it is possible that a parallel pathway may instead be affected. The exoribonuclease pacman is highly conserved in all eukaryotes, therefore it is likely that it is involved in similar morphological processes, such as wound healing in human cells.
核糖核酸酶在酵母和细菌中已得到充分研究，但它们对多细胞生物发育过程的生物学意义尚未完全了解。然而，越来越多的证据表明，特定时间的转录本降解对于许多细胞过程的调控至关重要，包括翻译抑制、无义介导的衰变和RNA干扰。果蝇基因pacman与主要的酵母外切核糖核酸酶XRN1高度同源，是真核生物中唯一已知的细胞质5'-3'外切核糖核酸酶。为了确定这种外切核糖核酸酶在发育中的作用，我们通过P元件切除在pacman中构建了一些突变，并对产生的表型进行了表征。
pacman中的突变导致果蝇出现许多特定表型，如活力低下、翅膀暗淡、腿部弯曲、背部/胸部闭合不正确以及伤口愈合缺陷。参与背部/胸部闭合的上皮细胞片运动是一个保守的形态发生过程，类似于脊椎动物后脑闭合和人类伤口愈合的过程。由于已知JNK（c-Jun N末端激酶）信号通路参与背部/胸部闭合和伤口愈合，我们测试了pacman是否影响JNK信号传导。我们的实验表明，pacman与puckered基因发生遗传相互作用，puckered是一种负调控JNK信号通路的磷酸酶。
这些结果表明，5'-3'外切核糖核酸酶pacman是果蝇上皮细胞片密封关键方面所必需的。由于这些突变导致特定表型，我们的数据表明外切核糖核酸酶Pacman靶向参与此过程的特定mRNA子集。其中一个靶点可能是JNK信号通路成员，尽管也有可能是平行通路受到影响。外切核糖核酸酶pacman在所有真核生物中高度保守，因此它可能参与类似的形态发生过程，如人类细胞中的伤口愈合。
