Ha I, Wightman B, Ruvkun G
Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
Genes Dev. 1996 Dec 1;10(23):3041-50. doi: 10.1101/gad.10.23.3041.
The Caenorhabditis elegans heterochronic gene lin-14 generates a temporal gradient of the LIN-14 proteins to control stage-specific patterns of cell lineage during development. Down-regulation of LIN-14 is mediated by the lin-14 3' untranslated region (UTR), which bears seven sites that are complementary to the regulatory lin-4 RNA. Here we report molecular and genetic evidence that RNA duplexes between the lin-4 and lin-14 RNAs form in vivo and are necessary for LIN-14 temporal gradient generation. lin-4 RNA binds in vitro to a lin-14 mRNA bearing the seven lin-4 complementary sites but not to a lin-14 mRNA bearing point mutations in these sites. In vivo, the lin-4 complementary regions are necessary for lin-14 3' UTR-mediated temporal gradient formation. Based on lin-14 3' UTR sequence comparisons between C. elegans and C. briggsae, four of the seven lin-4/lin-14 RNA duplexes are predicted to bulge a lin-4 C residue, and three sites are predicted to form nonbulged RNA duplexes. Reporter genes bearing multimerized bulged C lin-4 binding sites show almost wild-type temporal gradient formation, whereas those bearing multimerized nonbulged lin-4 binding sites do not form a temporal gradient. Paradoxically, lin-4 RNA binds in vitro to nonbulged lin-14 RNA more avidly than to the bulged lin-14 RNA. This suggests that a specific secondary structure of lin-4/lin-14 RNA duplex that may be recognized by an accessory protein, rather than an RNA duplex per se, is required in vivo for the generation of the LIN-14 temporal gradient.
秀丽隐杆线虫异时性基因lin-14产生LIN-14蛋白的时间梯度，以控制发育过程中细胞谱系的阶段特异性模式。LIN-14的下调由lin-14 3'非翻译区（UTR）介导，该区域有七个与调控性lin-4 RNA互补的位点。我们在此报告分子和遗传学证据，表明lin-4和lin-14 RNA之间的RNA双链体在体内形成，并且是LIN-14时间梯度产生所必需的。lin-4 RNA在体外与带有七个lin-4互补位点的lin-14 mRNA结合，但不与这些位点带有点突变的lin-14 mRNA结合。在体内，lin-4互补区域对于lin-14 3'UTR介导的时间梯度形成是必需的。基于秀丽隐杆线虫和布里格氏秀丽线虫之间的lin-14 3'UTR序列比较，预测七个lin-4/lin-14 RNA双链体中的四个会使lin-4 C残基形成凸起，三个位点预计会形成无凸起的RNA双链体。带有多聚化凸起C lin-4结合位点的报告基因显示出几乎野生型的时间梯度形成，而带有多聚化无凸起lin-4结合位点的报告基因则不形成时间梯度。矛盾的是，lin-4 RNA在体外与无凸起的lin-14 RNA结合比与有凸起的lin-14 RNA更 avidly。这表明，体内产生LIN-14时间梯度需要lin-4/lin-14 RNA双链体的特定二级结构，而不是RNA双链体本身，这种结构可能被一种辅助蛋白识别。
