Shaun Hunter Pasquinelli Lab Finding a better way to identify
targets of the let-7 miRNA in C elegans. Slide 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 Slide 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' Slide 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% 325 6 8 2 4 73 3 20 2 56 0 17 70 557 Target
Predictions Slide 5 Expression of let-7 miRNA is developmentally
regulated and correlates with disappearance of lin-41 mRNA during
development (Bagga et al. 2005) Slide 6 Expression of let-7 miRNA
is developmentally regulated and correlates with disappearance of
lin-41 mRNA during development (Bagga et al. 2005) Slide 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) Slide 8 122 up-regulated transcripts in the
let-7 mutant (3 independent experiments, p-values 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 Slide 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 Slide 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 Slide 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 Slide 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 Slide 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 Slide 22 RNAi testing: Up-regulated in let-7 (p
