Jones D, Candido E P
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, V6T 1Z3, Canada.
Dev Biol. 2000 Oct 1;226(1):152-65. doi: 10.1006/dbio.2000.9847.
This work has identified the enzymes involved in the activation and conjugation of the ubiquitin-like protein NED-8 in Caenorhabditis elegans. A C. elegans conjugating enzyme, UBC-12, is highly specific in its ability to utilize NED-8 as a substrate. Immunostaining shows that NED-8 is conjugated in vivo to a major target protein with a conjugate size of 90 kDa. While the amount of this conjugate is developmentally regulated with reduced levels in the larval stages, the mRNA encoding C. elegans UBC-12 is constitutively produced throughout development, as is NED-8 itself. The importance of the NED-8 conjugating system in C. elegans was determined by RNA interference (RNAi) assays using double-stranded RNA encoding NED-8, UBC-12, or the NED-8 activating enzyme component ULA-1. The progeny of both ned-8 and ubc-12 RNAi-treated hermaphrodites either arrested during embryonic development or underwent abnormal postembryonic development. The effect on postembryonic development was pleiotropic, the most frequent gross abnormality being vulval eversion during the L4 stage. Individuals with an everted vulva either burst at the L4 to adult molt or gave rise to adults incapable of egg laying. Additionally, both ned-8 and ubc-12 RNAi induced a striking abnormality in the alae, structures produced by the lateral hypodermal seam cells in the adult nematode. Affected alae were patchy and frequently diverged around a central space. Vulval defects were also produced by RNAi directed at C. elegans ula-1. This is the first demonstration of a requirement for NED-8 conjugation in metazoan development.
这项研究确定了秀丽隐杆线虫中参与类泛素蛋白NED - 8激活和缀合的酶。一种秀丽隐杆线虫缀合酶UBC - 12在利用NED - 8作为底物的能力方面具有高度特异性。免疫染色显示，NED - 8在体内与一种大小为90 kDa的主要靶蛋白缀合。虽然这种缀合物的量在发育过程中受到调控，在幼虫阶段水平降低，但编码秀丽隐杆线虫UBC - 12的mRNA在整个发育过程中持续产生，NED - 8本身也是如此。通过使用编码NED - 8、UBC - 12或NED - 8激活酶组分ULA - 1的双链RNA进行RNA干扰（RNAi）试验，确定了秀丽隐杆线虫中NED - 8缀合系统的重要性。经ned - 8和ubc - 12 RNAi处理的雌雄同体线虫的后代要么在胚胎发育期间停滞，要么经历异常的胚后发育。对胚后发育的影响是多效性的，最常见的明显异常是L4阶段的阴门外翻。阴门外翻的个体要么在L4到成虫蜕皮时破裂，要么产生不能产卵的成虫。此外，ned - 8和ubc - 12 RNAi均在成虫线虫的侧翼诱导了显著异常，侧翼是由侧皮下缝合细胞产生的结构。受影响的侧翼有斑块，并且经常围绕一个中央空间发散。针对秀丽隐杆线虫ula - 1的RNAi也产生了阴门缺陷。这是首次证明后生动物发育中NED - 8缀合的必要性。
