Spike Caroline A, Davies Andrew G, Shaw Jocelyn E, Herman Robert K
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis 55455, USA.
Development. 2002 Nov;129(21):4999-5008. doi: 10.1242/dev.129.21.4999.
Previous work has shown that C. elegans MEC-8 is a putative RNA-binding protein that promotes specific alternative splices of unc-52 transcripts. unc-52 encodes homologs of mammalian perlecan that are located extracellularly between muscle and hypodermis and are essential for muscle development in both embryos and larvae. We show that MEC-8 is a nuclear protein found in hypodermis at most stages of development and not in most late embryonic or larval body-wall muscle. We have also found that overexpression of MEC-8 in hypodermis but not muscle can suppress certain unc-52 mutant phenotypes. These are unexpected results because it has been proposed that UNC-52 is produced exclusively by muscle. We have constructed various tissue-specific unc-52 minigenes fused to a gene for green fluorescent protein that have allowed us to monitor tissue-specific mec-8-dependent alternative splicing; we show that mec-8 must be expressed in the same cell type as the unc-52 minigene in order to regulate its expression, supporting the view that MEC-8 acts directly on unc-52 transcripts and that UNC-52 must be synthesized primarily by the hypodermis. Indeed, our analysis of unc-52 genetic mosaics has shown that the focus of unc-52 action is not in body-wall muscle but most likely is in hypodermis.
先前的研究表明，秀丽隐杆线虫的MEC-8是一种假定的RNA结合蛋白，可促进unc-52转录本的特定可变剪接。unc-52编码哺乳动物基底膜聚糖的同源物，这些同源物位于肌肉和皮下组织之间的细胞外，对胚胎和幼虫的肌肉发育至关重要。我们发现，MEC-8是一种核蛋白，在发育的大多数阶段都存在于皮下组织中，而在大多数晚期胚胎或幼虫体壁肌肉中不存在。我们还发现，在皮下组织而非肌肉中过表达MEC-8可以抑制某些unc-52突变体表型。这些结果出乎意料，因为有人提出UNC-52仅由肌肉产生。我们构建了各种与绿色荧光蛋白基因融合的组织特异性unc-52微型基因，这些微型基因使我们能够监测组织特异性的mec-8依赖性可变剪接；我们表明，mec-8必须在与unc-52微型基因相同的细胞类型中表达才能调节其表达，这支持了MEC-8直接作用于unc-52转录本且UNC-52必须主要由皮下组织合成的观点。事实上，我们对unc-52基因嵌合体的分析表明，unc-52的作用焦点不在体壁肌肉中，而很可能在皮下组织中。
