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Shaun Hunter Pasquinelli Lab
Finding a better way to identify targets of the let-7 miRNA in C elegans. Shaun Hunter Pasquinelli Lab
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Regulation of specific miRNA primary transcripts
miRNA Pathway RISC (Argonaute Proteins) RNaseIII Dicer Maturation to 22nt miRNAs Regulation of specific mRNA targets ~22nt miRNAs Transcription of miRNA primary transcripts Processing to miRNA precursors Drosha
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Target Predictions Four prediction methods:
RNA22: pattern matching, 6-mer seed match PicTar: conservation, mfe cutoff, seed match Grosshans et al. 2005: some pairing 5’ and 3’, multiple sites Miranda: alignment score, seed bias, conservation target 5' U AUU U 3' UUAUACAACC CUGCCUC GAUAUGUUGG GAUGGAG miRNA 3' U AU U 5’ target 5' U GUU A A 3' UUAUACAACC CUAC CUCA GAUAUGUUGG GAUG GAGU miRNA 3' U AU '
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Target Predictions Four prediction methods:
RNA22: 425 total PicTar: 57 total Grosshans et al. 2005: 82 total Miranda: 660 total all find lin-41, daf-12, and hbl-1 Overlap of predictions poor 68% 35% 56 20 3 6 84% 76% 2 2 557 325 4 8 17 7 3 70
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Expression of let-7 miRNA is developmentally regulated and correlates with disappearance of lin-41 mRNA during development (Bagga et al. 2005)
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Expression of let-7 miRNA is developmentally regulated and correlates with disappearance of lin-41 mRNA during development (Bagga et al. 2005)
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Down-regulation of lin-41 mRNA is let-7 miRNA dependent
Northern L2 L4 let-7 (-) let-7 (-) WT WT lin-41 mRNA eft-2 mRNA (control) (-) (Bagga et al. 2005)
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122 up-regulated transcripts in the let-7 mutant
Differential gene expression in wild-type vs. let-7 mutants 122 up-regulated transcripts in the let-7 mutant (3 independent experiments, p-values <.01)
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Results Found known targets 675 probes up-regulated (p<0.01)
lin-41, hbl-1, nhr-25, daf-12 675 probes up-regulated (p<0.01) 58 genes overlap with previous predictions 108 probes up-regulated (p<0.001) 12 genes overlap with previous predictions
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Prioritizing Candidates
Northern Target properties Upregulated in let-7 L4 let-7 (-) WT lin-41 mRNA eft-2 mRNA (control)
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Prioritizing Candidates
Target properties Upregulated in let-7 RNAi against targets suppress let-7 phenotypes
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Suppression of bursting
Grow bacteria expressing dsRNA for RNAi against candidate gene Screen for suppression of let-7 phenotypes Analyze bursting percentage T7 Feed worms bacteria control candidate
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let-7 regulates late larval development
lin-41 larval genes Sulston & Horvitz, 1977 WT V1 L1 L2 L3 L4 Ad At the turn of the millennium we learned about another tiny RNA gene in C. elegans. Mutations in let-7 resulted in reiterations of late larval cell division patterns. This produces a number of abnormalities such as the rupturing seen in this poor worm which contribute to eventual lethality, hence the name.
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let-7 regulates late larval development
lin-41 larval genes Sulston & Horvitz, 1977 WT let-7 V1 L1 L2 L3 L4 Ad At the turn of the millennium we learned about another tiny RNA gene in C. elegans. Mutations in let-7 resulted in reiterations of late larval cell division patterns. This produces a number of abnormalities such as the rupturing seen in this poor worm which contribute to eventual lethality, hence the name. Reinhart et al., 2000
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let-7 regulates late larval development
lin-41 larval genes Sulston & Horvitz, 1977 WT let-7 Reinhart et al., 2000 V1 L1 L2 L3 L4 Ad At the turn of the millennium we learned about another tiny RNA gene in C. elegans. Mutations in let-7 resulted in reiterations of late larval cell division patterns. This produces a number of abnormalities such as the rupturing seen in this poor worm which contribute to eventual lethality, hence the name. N2 (wild-type) let-7 (mn112) lin-29 (n333)
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Suppression of Extra Seam Cell Nuclei
control candidate Grow bacteria expressing dsRNA for RNAi against candidate gene Feed worms bacteria Screen for suppression of let-7 phenotypes >16 seam nuclei < Control RNAi
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Prioritizing Candidates
Target properties Upregulated in let-7 RNAi against targets suppress let-7 phenotype Should be up-regulated in let-7 but not in lin-29 mutants
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Prioritizing Candidates
Wild-type larval genes Target properties Upregulated in let-7 RNAi against targets suppress let-7 phenotype Should be up-regulated in let-7 but not in lin-29 mutants let-7 lin-41 lin-29
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Prioritizing Candidates
Wild-type larval genes Target properties Upregulated in let-7 RNAi against targets suppress let-7 phenotype Should be up-regulated in let-7 but not in lin-29 mutants let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29
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Prioritizing Candidates
Wild-type larval genes Target properties Upregulated in let-7 RNAi against targets suppress let-7 phenotype Should be up-regulated in let-7 but not in lin-29 mutants let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29 lin-29 mutant larval genes let-7 lin-41 lin-29
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Prioritizing Candidates
Target properties Upregulated in let-7 RNAi against targets suppress let-7 phenotype Should be up-regulated in let-7 but not in lin-29 mutants
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Choosing RNAi test candidates
Wild-type larval genes RNAi testing: Up-regulated in let-7 (p<0.05) Different in lin-29 vs let-7 (p<0.05) Less up in lin-29 than let-7 ~200 genes Finished bursting screen (one week) let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29 let-7 lin-41 lin-29 larval genes lin-29 mutant
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Choosing RNAi test candidates
Wild-type larval genes RNAi testing: Up-regulated in let-7 (p<0.05) Different in lin-29 vs let-7 (p<0.05) Less up in lin-29 than let-7 ~200 genes Finished bursting screen (one week) let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29 let-7 lin-41 lin-29 larval genes lin-29 mutant
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suppression
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Bursting supressors ZC247.3 W05B10.5 F28C1.1
lin-11, TF involved in vulval development W05B10.5 srx-116 7TM receptor, Ste/Emb F28C1.1 SWAP mRNA splicing regulator F45F2.12 /// F07B7.4 /// F07B7.11 /// K06C4.12 /// K06C4.4 H2B histones F08C6.1 Metallo-protease Unc, Lva, Dpy F53F4.5 fmo-4 flavin mono-oxygenase C26E6.6 Ribosomal protein Lva, Lvl, Ste T08B2.8 MT ribosomal protein Lva, Emb F42A8.1 Unknown nematode only Lva, Dpy, Ste/Emb F45D3.4 Unknown nematode only f59e11.7
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Validating candidates using a reporter
GFP::lin-41 UTR (+LCS) GFP::lin-41 UTR ∆LCS
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Reporter expression regulated by lin-41 UTR
ap124 L2 L4 ap128 L2 L4 ap129 L2 L4 N2 L2 L4 let-7 L2 L4 ap119 L2 L4 ap120 L2 L4 ap121 L2 L4 ap123 L2 L4 N2 L2 L4 let-7 L2 L4 lin-41 lin-41 GFP GFP actin actin 28S rRNA 28S rRNA ap122 L2 L4 ap125 L2 L4 ap127 L2 L4 ap143 L2 L4 ap144 L2 L4 GFP actin 28S rRNA
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Regulation is LCS dependent
ap145 L2 L4 ap146 ap150 ap151 ap152 ap153 lin-41 ap122 L2 L4 ap125 ap127 ap143 ap144 GFP actin 28S rRNA GFP GFP actin 28S rRNA
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Regulation is sensitive to expression level
5 4.5 4 3.5 3 Fold change (L2/L4) 2.5 2 1.5 1 0.5 + LCS UTR Low -- LCS UTR Low + LCS UTR High -- LCS UTR High
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Constructs with functional binding sites are regulated at the mRNA and protein levels
Northern Western + LCS -- LCS daf-12
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Candidate testing status
Injecting 5 constructs at a time positive control, negative control, and three test UTRs First six to be tested nhr-25, nhr-71, ztf-7, col-90, F41E6.14, T14B1.1 Have RNA from 3 lines from one mix of candidates, and 4 from the other ztf-7 and nhr-71 appear regulated
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ztf-7 and nhr-71 UTRs appear to be regulated
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For many genes the endogenous seems to be the bulk of signal
col-90 F41E6.14 T14B1.1
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Timetable Screen for seam cell phenotype suppression
~2-3 weeks Cloning of new candidate UTRs (~30) ~1 month Injection and isolation of transgenic lines ~2-3 months Northern analysis and/or qRT-PCR
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Acknowledgements Genechip Core Gene Yeo (Salk) Bioinformatics UCSD Center for AIDS Research, Genomics Core qRT-PCR Amy Pasquinelli Pasquinelli Lab Shveta Bagga John Bracht Katlin Massirer Janette Holtz Brad Hehli Zoya Kai Brian Maurer UCSD Center for AIDS Research, Genomics Core (qRT-PCR)
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