microrna pseudo-targets

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1 microrna pseudo-targets Natalia Pinzón Restrepo Institut de Génétique Humaine (CNRS), Montpellier, France August, 2012

2 microrna target identification mir: target: N NNNNNNNNNNNNNN NNNNNN 3 the seed

3 Most Identification of mirna targets computational programs for target prediction: search seed matches in 3 UTRs, select the ones that were conserved in evolution

4 Most Identification of mirna targets computational programs for target prediction: search seed matches in 3 UTRs, select the ones that were conserved in evolution Such short matches are very frequent (60 % of human coding genes seem to be targeted: Friedman et al, 2009)

Most Identification of mirna targets computational programs for target prediction: search seed matches in 3 UTRs, select the ones that were conserved in evolution Such

5 Most Identification of mirna targets computational programs for target prediction: search seed matches in 3 UTRs, select the ones that were conserved in evolution Such short matches are very frequent (60 % of human coding genes seem to be targeted: Friedman et al, 2009) = mirnas are implicated in every physiological process in animals

6 Paradox mirna-mediated repression is very modest (usually < 2-fold) (Baek et al, 2008, Selbach et al, 2008), but biological processes are robust: they can tolerate considerable fluctuations in parameters (such as genetic variation) and still generate invariant phenotypic outputs

7 Paradox mirna-mediated repression is very modest (usually < 2-fold) (Baek et al, 2008, Selbach et al, 2008), but biological processes are robust: they can tolerate considerable fluctuations in parameters (such as genetic variation) and still generate invariant phenotypic outputs = how mirnas accomplish any significant regulation (a small change in gene expression triggers a physiological effect)?

8 An alternative hypothesis

9 An alternative hypothesis Most computationally predicted targets are not functionally targeted (not repressed enough) Their phylogenetic conservation means that these binding sites have a function pseudo-target (insensitive)

10 An alternative hypothesis Most computationally predicted targets are not functionally targeted (not repressed enough) Their phylogenetic conservation means that these binding sites have a function That function could be to repress the mirna by titrating it pseudo-target (insensitive) real target (sensitive)

11 Discriminative prediction 1 mrna microrna mrna According to the new hypothesis: mirna binding sites should be better conserved in abundantly expressed pseudo-targets

12 Discriminative prediction 1 mrna microrna mrna According to the new hypothesis: mirna binding sites should be better conserved in abundantly expressed pseudo-targets According to the current theory: mirna binding sites conservation is not expected to correlate with gene expression

13 Are seed match conservation and mrna abundance correlated?

14 Are seed match conservation and mrna abundance correlated? mirna binding site conservation: measured by TargetScan s P CT score (Friedman et al, 2009) Quantification of mrna abundance: extracted from published microarray experiments (Mackiewicz et al, 2007, Thorrez et al, 2008)

15 Are seed match conservation and mrna abundance correlated? Kendall's τ = (p-value = ) Conservation score (P CT ) of binding sites to mir Gene expression in hypothalamus

16 Are seed match conservation and mrna abundance correlated? Kendall's τ = (p-value = ) Conservation score (P CT ) of binding sites to mir Gene expression in hypothalamus abundantly expressed mir-17 targets tend to bear highly conserved binding sites

17 Are seed match conservation and mrna abundance correlated? hypothalamus Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 mir Kendall s τ

18 Are seed match conservation and mrna abundance correlated? hypothalamus kidney Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 mir-17 Adjusted p value 1e 16 1e 12 1e 08 1e Kendall s τ Kendall s τ +34 other mouse tissues, same results: Supplementary data +33 fly tissues, same results: Supplementary data

19 Discriminative prediction 2 According to the current theory: mirna targets are tightly regulated (inter-individual fluctuation should not exceed mirna-guided repression)

20 Discriminative prediction 2 According to the current theory: mirna targets are tightly regulated (inter-individual fluctuation should not exceed mirna-guided repression) According to the new hypothesis: Pseudo-target expression levels can fluctuate between individuals in a natural population (phenotype is robust)

21 Natural variability vs mirna-guided repression

22 Natural variability vs mirna-guided repression Baek et al, 2008: quantification of mir-223-mediated repression in mouse neutrophils

23 Natural variability vs mirna-guided repression Baek et al, 2008: quantification of mir-223-mediated repression in mouse neutrophils Blood collection Neutrophil isolation RNA extraction cdna labeling, array hybridization

24 Natural variability vs mirna-guided repression Baek et al, 2008: quantification of mir-223-mediated repression in mouse neutrophils Blood collection Neutrophil isolation Blood collection Pooled blood Split in 5 replicates Neutrophil isolation RNA extraction RNA extraction cdna labeling, array hybridization cdna labeling, array hybridization

25 Natural variability vs mirna-guided repression

26 Natural variability vs mirna-guided repression

27 Natural variability vs mirna-guided repression

28 Natural variability vs mirna-guided repression p: probability that the difference between two individual mice is smaller than mirna-guided repression

29 Natural variability vs mirna-guided repression For 168 predicted targets out of 189: inter-individual fluctuations across 5 wild-type mice exceeds mirna-mediated regulation (p-value < 005)

30 Natural variability vs mirna-guided repression For 168 predicted targets out of 189: inter-individual fluctuations across 5 wild-type mice exceeds mirna-mediated regulation (p-value < 005) the remaining 21 targets:

31 Conclusion: revisiting mirna target definition

32 Conclusion: revisiting mirna target definition Every measurable change in gene expression does not translate into a macroscopic, evolutionarily selectable phenotype gene expression phenotype

33 Acknowledgements Hervé Seitz, Anna Sergeeva, and Laura Martinez Jessy Presumey and Florence Apparailly (INM, Montpellier, France)

34 Supplementary data Alternative interpretations of properties of predicted mirna targets Conservative approximations in the assessment of abundance/conservation correlation Positive correlation between gene expression and conservation of mirna binding sites Dose-sensitivity and seed match conservation

35 Alternative interpretations of properties of predicted mirna targets mrna for gene 1 mrna for gene 1 mrna for gene 2 mrna for gene 2 mirna mirna mrna for gene 3 mrna for gene 3 mrna for gene 4 mrna for gene 4 Return

36 Alternative interpretations of properties of predicted mirna targets mirna mrna mirna and mrna expression mrna sharp boundary of mrna activity domain mirna and mrna expression mirna sharp boundary of mirna activity domain spatial or temporal axis spatial or temporal axis Return

37 Alternative interpretations of properties of predicted mirna targets mirna mrna for tissue specific gene mirna mrna for tissue specific gene avoidance avoidance cell type 1 mrna for house keeping gene cell type 1 mrna for house keeping gene mirna mrna for tissue specific gene mirna mrna for tissue specific gene avoidance avoidance cell type 2 mrna for house keeping gene cell type 2 mrna for house keeping gene Return

38 mrna abundance and seed match conservation Most predicted targets are expected to be pseudo-targets (conservative approximation: we will consider every predicted target) mirna binding site should correlate with that mrna s abundance in the cells where mirna titration is beneficial (conservative approximation: we will consider whole tissues and organs) Poorly abundant mrnas (without a real titration effect on the mirna) should not exhibit such correlation (conservative approximation: we will consider every mrna) Return

39 mrna abundance and seed match conservation splenic B cells spleen naive B cells Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e Kendall s τ Kendall s τ Kendall s τ testis ES cells ovary Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e Kendall s τ Kendall s τ Kendall s τ Return

40 mrna abundance and seed match conservation adult ovary adult eye adult heart Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e Expression of deeply conserved/expression of poorly conserved Expression of deeply conserved/expression of poorly conserved Expression of deeply conserved/expression of poorly conserved adult hind gut adult salivary gland larval feeding fatbody Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 Adjusted p value 1e 16 1e 12 1e 08 1e Expression of deeply conserved/expression of poorly conserved Expression of deeply conserved/expression of poorly conserved Expression of deeply conserved/expression of poorly conserved Return

41 mrna abundance ans seed match conservation hypothalamus Adjusted p value 1e 16 1e 12 1e 08 1e 04 1 mir-122 mir-150 mir-124 mir-138 mir-9 mir-181a mir-1a mir-133 neuron-specific micrornas non neuron-specific micrornas Kendall s τ Return

42 Return Dose-sensitivity and seed match conservation

robustness: revisting the significance of mirna-mediated regulation

robustness: revisting the significance of mirna-mediated regulation : revisting the significance of mirna-mediated regulation Hervé Seitz IGH (CNRS), Montpellier, France October 13, 2012 microrna target identification .. microrna target identification mir: target: 2 7