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First published online 25 October 2006
doi: 10.1242/dev.02655

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The mir-84 and let-7 paralogous microRNA genes of Caenorhabditis elegans direct the cessation of molting via the conserved nuclear hormone receptors NHR-23 and NHR-25

Department of Genetics, Harvard Medical School and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.

View larger version (48K): [in a new window]   Fig. 1. Genetic analysis of mir-84. (A) Sequence alignment of the let-7 family members of C. elegans. Residues identical between mir-84 and let-7 are shown in bold. Boxes indicate residues conserved among all let-7 paralogs. (B) The mir-84 genomic region. Numbers correspond to cosmid B0395 (Accession number: emb|Z68131). The dashed line indicates the extent of deletion tm1304. Arrows indicate mature mir-84 and related primers. (C,D) Northern blots showing mir-84 levels in early L4-stage larvae of the indicated genotypes. A shorter exposure time was used in D than in C. Levels of 5S rRNA, stained by ethidium bromide, or U6 snRNA, provide loading controls.

View larger version (42K): [in a new window]   Fig. 2. mir-84 acts synergistically with let-7 to promote the cessation of molting. (A) Nomarski image of a let-7(mg279) mir-84(tm1304) adult. The arrow indicates partly shed cuticle. (B) Inviability of adults of the indicated genotypes.

View larger version (40K): [in a new window]   Fig. 3. let-7 mir-84 adults express gfp reporters associated with molting. (A-D) Animals were cultivated at 25°C following release from starvation at the early L1 stage. (A) Prevalence of fluorescence from mlt-10p::gfp-pest, as detected with a Zeiss SV-6 microscope. Each sample contained 52 or more animals. Data were collected in three experiments to cover all time points. Error bars indicate the standard deviation from the mean in cases where two populations of animals were observed at the same time point. (B) Fluorescence from mlt-10p::gfp-pest in adults cultivated for 56 hours. (C) Western blot showing GFP in protein extracts from late L4-stage or gravid adults cultivated, respectively, for 41 or 56 hours. Each strain carried the mgIs49[mlt-10p::gfp-pest] transgene. Levels of ß-tubulin provide a loading control. (D) Fluorescence from nas-37p::gfp-pest in adults.

View larger version (9K): [in a new window]   Fig. 4. Inactivation of precocious heterochronic genes prevents the supernumerary molt of let-7 mir-84 mutants. Synchronized populations of L1 larvae were fed bacteria expressing dsRNA corresponding to the indicated genes and cultivated at 25°C. Animals in the P0 generation were scored for fluorescence from mlt-10p::gfp-pest 56 hours later and then for viability the next day. To score F1 animals, progeny were collected, synchronized as L1 larvae, and then fed the appropriate bacterial clone for an additional 55 hours.

View larger version (22K): [in a new window]   Fig. 5. Inactivation of nhr-23 or nhr-25 blocks the supernumerary molt of let-7 mir-84 mutants. (A,B) Synchronized populations of let-7(mg279) mir-84(tm1304) animals were cultured until the fourth larval stage at 20°C. Individuals were then fed bacteria expressing dsRNA and observed several times over the next 2 days. The combined results of three independent experiments are shown. Asterisks indicate a significant difference from the control sample (P 0.001, chi-square test). (A) Prevalence of detectable fluorescence from mlt-10p::gfp-pest. (B) Prevalence of death. (C) Fluorescence from nhr-23::gfp in the nuclei of epithelial cells near the head of let-7 mir-84 or wild-type adults.

View larger version (24K): [in a new window]   Fig. 6. Potential binding sites for mir-84 and paralogs in the 3' UTR of nhr-25. (A) The 3' UTR of C. elegans nhr-25, corresponding to nucleotides 35690 through 36438 of cosmid F11C1 (Accession number: emb|Z54270). Lines denote potential binding sites for let-7 and related miRNAs, as indicated. (B) Vertical lines indicate potential base-pairing between the 3' UTR of C. elegans nhr-25 and the let-7-like miRNA, for each site shown in A. The predicted free energy of folding is indicated. Sequence alignments show conservation of the putative binding sites in the 3' UTRs of predicted nhr-25 orthologs from C. briggsae (Accession number: emb|CAAC01000078) and C. remanei (contig 0.60, nucleotides 49497 through 50100). Asterisks indicate nucleotides perfectly conserved among all three species, including seed sequences for the predicted mir-84 and let-7 sites. Colons indicate substitutions within seed sequences of the predicted mir-48 and mir-241 binding site compatible with G:U base-pairing.

View larger version (67K): [in a new window]   Fig. 7. Expression of mir-84 fusion genes. (A) Fluorescence from mir-84::gfp in the seam cells of an L4-stage larva. (B) Fluorescence from mir-84::gfp in the seam cells, pharynx and somatic gonad of an L2-stage larva. (C) Fluorescence from mir-84::yfp in the seam cells of an L3-stage larva. Arrowheads indicate seam cells.

View larger version (8K): [in a new window]   Fig. 8. mir-84 and let-7 promote full expression of col-19::gfp in adults. Loss of mir-84 exacerbates the failure of let-7(mg279) mutants to express col-19::gfp in both the hypodermis and seam cells. Gravid adults in mixed-stage populations were scored using a Zeiss SV-6 microscope.

View larger version (3K): [in a new window]   Fig. 9. Overexpression of mir-84 rescues a null allele of let-7. Graph shows the percent of let-7(mn112) animals that survived and produced progeny in the presence or absence of mgIs45[mir-84++]. We compared GR1426 to animals derived from SP231 that displayed uncoordinated movement.

View larger version (4K): [in a new window]   Fig. 10 . A genetic model for the cessation of molting. We propose that let-7 and mir-84 act through the heterochronic pathway to repress key regulators of molting, including the conserved nuclear hormone receptor genes nhr-23 and nhr-25. let-7 and paralogous miRNAs might also target nhr-25 mRNA. Positive regulation is denoted by an arrowhead and negative regulation by a perpendicular line.