A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation.
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.
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Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
Wightman
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B
Ruvkun
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G
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000196:embryology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins; D017124:Conserved Sequence; D015536:Down-Regulation; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017354:Point Mutation; D014176:Protein Biosynthesis; D018510:RNA, Helminth / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D015801:Helminth Proteins; C102669:LIN-14 protein, C elegans; D009687:Nuclear Proteins; D018510:RNA, Helminth