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Finding a better way to identify targets of the let-7 miRNA in C elegans.

Finding a better way to identify targets of the let-7 miRNA in C elegans. Shaun Hunter Pasquinelli Lab. RNaseIII. RNaseIII. Drosha. Processing to miRNA precursors. Transcription of miRNA primary transcripts. Maturation to 22nt miRNAs. RNaseIII. RNaseIII. Dicer. RISC (Argonaute

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Finding a better way to identify targets of the let-7 miRNA in C elegans.

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  1. Finding a better way to identify targets of the let-7 miRNA in C elegans. Shaun Hunter Pasquinelli Lab

  2. RNaseIII RNaseIII Drosha Processing to miRNA precursors Transcription of miRNA primary transcripts Maturation to 22nt miRNAs RNaseIII RNaseIII Dicer RISC (Argonaute Proteins) Regulation of specific mRNA targets ~22nt miRNAs miRNA Pathway

  3. 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 5'

  4. 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 0 6 84% 76% 2 2 557 325 4 8 17 7 3 70

  5. Expression of let-7 miRNA is developmentally regulated and correlates with disappearance of lin-41 mRNA during development (Bagga et al. 2005)

  6. Expression of let-7 miRNA is developmentally regulated and correlates with disappearance of lin-41 mRNA during development (Bagga et al. 2005)

  7. 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)

  8. 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)

  9. Results • Found known targets • 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

  10. Prioritizing Candidates Target properties • Upregulated in let-7 Northern L4 let-7 (-) WT lin-41 mRNA eft-2 mRNA (control)

  11. Prioritizing Candidates Target properties • Upregulated in let-7 • RNAi against targets suppress let-7 phenotypes

  12. control candidate Grow bacteria expressing dsRNA for RNAi against candidate gene T7 T7 Feed worms bacteria T7 T7 Screen for suppression of let-7 phenotypes Analyze bursting percentage Suppression of bursting

  13. WT L1 V1 L2 L3 L4 Ad Sulston & Horvitz, 1977 let-7 regulates late larval development let-7 lin-41 larval genes

  14. WT L1 V1 L2 L3 L4 Ad Sulston & Horvitz, 1977 let-7 regulates late larval development let-7 lin-41 larval genes let-7 Reinhart et al., 2000

  15. let-7 WT L1 V1 L2 L3 L4 Ad Sulston & Horvitz, 1977 Reinhart et al., 2000 let-7 regulates late larval development let-7 lin-41 larval genes N2 (wild-type) let-7 (mn112) lin-29 (n333)

  16. T7 T7 T7 T7 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

  17. 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

  18. Prioritizing Candidates Wild-type 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 larval genes let-7 lin-41 lin-29

  19. Prioritizing Candidates Wild-type 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 larval genes let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29

  20. Prioritizing Candidates Wild-type 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 larval genes 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

  21. 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

  22. lin-29 mutant larval genes let-7 lin-41 lin-29 Choosing RNAi test candidates Wild-type • 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) larval genes let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29

  23. lin-29 mutant larval genes let-7 lin-41 lin-29 Choosing RNAi test candidates Wild-type • 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) larval genes let-7 lin-41 lin-29 let-7 mutant larval genes let-7 lin-41 lin-29

  24. suppression

  25. Bursting supressors • ZC247.3 • 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 • Unknown nematode only

  26. Validating candidates using a reporter GFP::lin-41 UTR (+LCS) GFP::lin-41 UTR ∆LCS

  27. Reporter expression regulated by lin-41 UTR ap119 L2 L4 ap120 L2 L4 ap121 L2 L4 ap123 L2 L4 N2 L2 L4 let-7 L2 L4 ap124 L2 L4 ap128 L2 L4 ap129 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

  28. ap145 L2 L4 ap146 L2 L4 ap150 L2 L4 ap151 L2 L4 ap152 L2 L4 ap153 L2 L4 ap122 L2 L4 ap125 L2 L4 ap127 L2 L4 ap143 L2 L4 ap144 L2 L4 GFP actin 28S rRNA Regulation is LCS dependent lin-41 GFP GFP actin 28S rRNA

  29. 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 0 + LCS UTR Low -- LCS UTR Low + LCS UTR High -- LCS UTR High

  30. Constructs with functional binding sites are regulated at the mRNA and protein levels Northern Western + LCS -- LCS daf-12

  31. 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

  32. ztf-7 and nhr-71 UTRs appear to be regulated nhr-25 nhr-71 ztf-7

  33. For many genes the endogenous seems to be the bulk of signal col-90 F41E6.14 T14B1.1

  34. 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 ~2-3 months

  35. Amy Pasquinelli Pasquinelli Lab Shveta Bagga John Bracht Katlin Massirer Janette Holtz Brad Hehli Zoya Kai Brian Maurer Genechip Core Gene Yeo (Salk) Bioinformatics UCSD Center for AIDS Research, Genomics Core qRT-PCR Acknowledgements • UCSD Center for AIDS Research, Genomics Core (qRT-PCR)

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