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

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Presentation on theme: "Shaun Hunter Pasquinelli Lab Finding a better way to identify targets of the let-7 miRNA in C elegans."— Presentation transcript:

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

2 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 RNaseIII Drosha 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 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 76% 68% 84% 35% Target Predictions

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 WT let-7 (-) WT let-7 (-) L4L2 Northern lin-41 mRNA eft-2 mRNA (control) (-) (Bagga et al. 2005)

8 122 up-regulated transcripts in the let-7 mutant (3 independent experiments, p-values <.01) Differential gene expression in wild-type vs. let-7 mutants

9 Results Found known targets –lin-41, hbl-1, nhr-25, daf 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 WT let-7 (-) L4 Northern lin-41 mRNA eft-2 mRNA (control)

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

12 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 controlcandidate

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

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

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

16 Grow bacteria expressing dsRNA for RNAi against candidate gene Screen for suppression of let-7 phenotypes T7 Feed worms bacteria controlcandidate >16 seam nuclei < Control RNAi Suppression of Extra Seam Cell Nuclei

17 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 Prioritizing Candidates

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

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

20 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-7lin-41lin-29 larval genes Wild-type let-7lin-41lin-29 larval genes let-7 mutant let-7lin-41lin-29 larval genes lin-29 mutant Prioritizing Candidates

21 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 Prioritizing Candidates

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

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

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 N2 L2 L4 let-7 Reporter expression regulated by lin-41 UTR lin-41 GFP actin 28S rRNA ap119 L2 L4 ap120 ap121 ap123 ap124 ap128 ap129 N2 let-7 ap122 ap125 ap127 ap143 ap144 GFP actin 28S rRNA lin-41 GFP actin 28SrRNA

28 ap122 L2 L4 ap125 ap127 ap143 ap144 GFP actin 28S rRNA Regulation is LCS dependentap145 L2 L4 ap146 ap150 ap151 ap152 ap153 lin-41 GFP actin 28S rRNA GFP

29 Regulation is sensitive to expression level Fold change (L2/L4) + LCS UTR Low + LCS UTR High -- LCS UTR Low -- LCS UTR High

30 Constructs with functional binding sites are regulated at the mRNA and protein levels NorthernWestern + 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 UCSD Center for AIDS Research, Genomics Core (qRT- PCR) Acknowledgements 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


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