microrna Shifra Ben-Dor March 2010

Transcription

1 microrna Shifra Ben-Dor March 2010

2 Outline Biology of mirna Prediction of mirna mirna Databases Prediction of mirna Targets

3 micrornas (mirna) Naturally expressed small RNAs Involved in regulation of target gene expression, in various ways: Repression of translation Cleavage of mrna In cases of quiescence, can promote translation

4 micrornas (mirna) Generally cause fine tuning of protein levels an efficient individual mir can knockdown protein expression by ~30% Most genes are under regulation by mir, and many have more than one binding site More binding sites = more knockdown Sites within ~40-50 bp of each other act synergistically, others independently

5 Biogenesis of mirna mir genes are found in several forms: Individual genes, complete with promoter (most PolII, some PolIII) Polycistronic genes with several mir Inside other genes, generally in introns Genes encode long ssrna molecules which form a hairpin loop and a region of complimentary binding

6 Biogenesis of mirna Stem-loops are processed into mature molecules that are nucleotides long (in plants can be up to ~24) Figure from: David Bartel Cell 116: (2004)

7 The mirna biogenesis pathway Du, T. et al. Development 2005;132:

8 Taken from: Pei and Tuschl, Nat Methods Sep;3(9):670-6!

9 microrna (mirna)! Processed into molecules called mirna duplexes An asymmetry in the thermodynamics of the 5 and 3 ends of the mirna duplex results in preferential loading of one strand into the RISC/ mirnp complex It was originally thought that the passenger strand, also known as mirna*, was usually degraded. Its now known that it is more active than originally shown

10 microrna (mirna)! mir bind by complementarity to transcripts of expressed genes. In mammals, there is generally not full complementarity, though in plants it is usually full or almost full. The more there is complementarity, the more the mrna gets cleaved Plant mirnas can bind anywhere in the transcript, and more are found in the CDS Most mammalian mirnas characterized to date bind to 3 untranslated regions of transcript mrnas

11 microrna (mirna)! Most mammalian mirnas characterized to date bind to 3 untranslated regions of transcript mrnas: Preferentially out of the way of the ribosome At least 15 bp after the stop codon Towards the ends of long UTRs, as opposed to the middles (possibly due to secondary structure of UTR) Preference for AU rich regions

12 microrna (mirna)! Many types of binding have been found. The major determinants are: The seed - bases 2-7 An A in position 1 of the target, with a mismatch in the mir Additional 3 binding

13 Increasing efficacy Offset 6mer site...nnnnnn... mrna 6mer site...nnnnnn... 7mer-A1 site...nnnnnna... 7mer-m8 site...nnnnnnn... 8mer site...nnnnnnna... NNNNNNNNNNNNNNNNNNNNN-5 mirna Seed Friedman et al. Genome Research :92-105

14 Friedman et al. Genome Research :92-105

15 Bartel, Cell :

16 mir families micrornas with the same seed are considered related, and are grouped into families Nomenclature can be tricky usually a mir with a, b, c...afterwards are family members, but there also can be family members with different numbers

17 What can we do bioinformatically with mirs? We can look for microrna genes We can look for microrna targets

18 How do we predict mirnas? Precursors form stable stem-loop structures, with continuous helical pairing and a few internal bulges mirnas are usually highly conserved among the genomes of related species Evolutionary divergence between orthologous mirnas shows a characteristic pattern: the terminal loops usually have more mutations than the arms of the stem-loops, and the mirna coding arms are more conserved than the nonmirna coding ones. Taken from: Geno. Prot. Bioinfo. Vol 3 No p

19 How do we predict mirnas? Structure (stem-loop) Conservation Non-coding Training and Test sets In nematodes, there is a conserved upstream element

20 Where can we find them? mirbase - all organisms, computational and experimental most comprehensive mirwalk (formerly Argonaute) - mammalian only (human, mouse, rat), Tarbase - experimentally supported, multiple organisms (includes plants) mirnamap

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22 How do we predict targets? In plants, its relatively easy - almost full complementarity In others we look for: Seed match (of all types) Evolutionary conservation Multiple sites Positioning of sites Free energy of mir-target duplex Accessibility of target Co-expression of mir and target

23 Hammell M. Computational methods to identify mirna targets. Semin Cell Dev Biol (2010), doi: /j.semcdb

24 Problems with these methods Most only check for 3 UTR sites Where do they get their 3 UTR collections from?? Problems with 3 UTR collections : Gene Models RefSeq Alternates

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26 Problems with these methods Conservation issues: prealigning vs any hit how far do we look at conservation? mammalian? vertebrate? How important is conservation for the less conserved mirs? Thermodynamics are tough to model for a full length 3 UTR Co-expression issues

27 How reliable are the predictions? Most of our data come from a little information on the most highly expressed mirs Most experiments were done on overexpression/knockdown systems Cell/Species specificity Methods of validation Genomics vs transcriptomics vs proteomics

28 Which program should I use? That depends our your biological question: What mirs regulate my gene? What genes are regulated by my mir? What genes are regulated by my mirs? Where is my mir expressed? How can I combine mir and mrna microarray data? Are there any common pathways affected by the same mir/combination of mirs?

29 Hammell M. Computational methods to identify mirna targets. Semin Cell Dev Biol (2010), doi: /j.semcdb

30 Bartel, Cell :

31 Validation tip Most cloned genes don t have their native 3 UTR - you use whats in the plasmid.

32 Links from this talk can be found: Toolbox Sequence Analysis by Target RNA mirna

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