micro RNA

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micro RNAs
Computational biology seminar Ariel Jaimovich
November 17th 2005
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The central dogma of biology

• This is not always the case
• First life forms
• viruses

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Dioxy ribo _at__at_?!
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Rna performs many functions

• Ribosomes
• tRNA
• Nuclear detaining
• RNAi

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Micro RNA

• 22nt rna
• Precursor stem loop
• Post-transcription regulation

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(No Transcript)
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miRNA History

• Lin-4 inhibits LIN14, but no LIN 4 protein was
  found (1993)

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miRNA appear in many organisms

• Highly conserved, many copies in each organism
• 4 paralogs of let7 4 in c elegans
• 15 in human
• 1 in drosophila

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miRNA Genes

• 1/3 Reside inside introns
• 2/3 independent transcription units
• Often in clusters.
• Many times near the genes they regulate or inside
  them.

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Expression

• Stage\tissue specific
• Large number of copies (robust transcription \
  slow decay)

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miRNA biogenesis
Highly conserved in evolution
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Plants Vs Animals
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miRNA - function
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Sequence recognition

• Positions 2-8 are most important
• How do we know
• Why ?

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Base pairing ? Function
How do we know which process is active ?
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Function (cont)

• Plants vs animals
• Number of target seq. on 3 utr ?
• Some miRNA target the same mRNA in different sites

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siRNA vs miRNA

• Genomic origin
• miRNA from genes
• siRNA from mRNAs, transposons, viruses...
• Synthesis
• One siRNA duplex ? many siRNA
• Conservation

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miRNA vs siRNA
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miRNA in plants

• Near-perfect complementarity
• mRNA cleavage, usually of TFs related to
  developmental processes
• Conservation between Arabidopsis and rice
• Defend against viruses

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miRNA in animals

• mRNA cleavage or translational silencing
• Conservation is also high (?)
• Different numbers of paralogs

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• Identification of hundreds of conserved and
  nonconserved human microRNAs
• Isaac Bentwich, Amir Avniel, Yael Karov, Ranit
  Aharonov Shlomit Gilad, Omer Barad, Adi
  Barzilai, Paz Einat, Uri Einav, Eti Meiri, Eilon
  Sharon, Yael Spector  Zvi Bentwich
• Nature genetics - June 2005

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Goal

• Find new human micro RNAs

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Motivation

• Current gene search techniques
• Hairpins
• Conservation

Try to search with a wider scope
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Search algorithm
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Prediction (1)
Fold the genome
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Magic box

• Structure features
• Hairpin length
• Loop length
• Stability score
• Free energy per nucleotide
• Matching pairs
• Bulge size

• Sequence features
• Sequence repetitiveness
• Regular internal repeat
• Inverted internal repeat

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Prediction (2)
430,000 hairpins
sample
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Prediction (3)
800 clustered
3000 non-clustered
1500 clustered
7500control
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Prediction (3)
69 adjacent miRNA
359 miRNA
Validate (clone and sequence)
89 (33 adjacent) cloned and sequenced NEW miRNA

• Of these
• 1 from the control list
• 36 conserved miRNAs (32 validated in other
  experiments)
• 43 in two new clusters

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New cluster
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The cluster conatin a few seeds
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Results summary
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(No Transcript)
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Goal
Location on chromosome
Expression
?
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Creating miRNA micro array
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Design microRNA chip

• Normalization by synthetic samples
• Melting temperature

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Array Results
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Is Expression correlated with distance between
microRNAs?
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Is expression of micro RNAs correlated with host
genes ?
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Caveats

• Numbers of pairs ?
• Quantitative comparison with host genes

Conclusions

• Some miRNA are arranged in genes
• miRNA that are located inside introns are
  expressed similarly to their hosts

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Points for thought

• Is miRNA regulated ? On which levels ?
• Is there a regulation on the RISC loading
• Why is so many annotated miRNA related to
  differentiation ?
• mRNA can be passed on during mitosis and need to
  cleaved
• Control leaky transcription ?