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

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

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'

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

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

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

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)

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)

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

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

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

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

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.

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

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)

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

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

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

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

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

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

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

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

suppression

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

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

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

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

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

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

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

ztf-7 and nhr-71 UTRs appear to be regulated

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

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

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)