Download MicroRNAs

micro RNA’s
Computational biology seminar
Ariel Jaimovich
November 17th 2005
The central dogma of biology
Transcription
Translation
RNA
• This is not always the case:
– First ‘life forms’
– viruses
Protein
Dioxy ribo @#$%##@?!
Rna performs many functions
•
•
•
•
Ribosomes
tRNA
Nuclear detaining
RNAi
Micro RNA
• ~22nt rna
• Precursor stem&
loop
• Post-transcription
regulation
miRNA History
• Lin-4 inhibits LIN14, but no LIN 4 protein
was found (1993)
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
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.
Expression
• Stage\tissue specific
• Large number of copies
(robust transcription \
slow decay)
miRNA – biogenesis
Highly conserved in evolution
Plants Vs Animals
miRNA - function
Sequence recognition
• Positions 2-8 are most important
– How do we know
– Why ?
Base pairing  Function
How do we know which process is active ?
Function (cont)
• Plants vs animals
• Number of target seq. on 3’ utr ?
• Some miRNA target the same mRNA in
different sites
3’ utr
Protein
coding rna
siRNA vs miRNA
• Genomic origin –
– miRNA from genes
– siRNA from mRNAs, transposons,
viruses...
• Synthesis
• One siRNA duplex  many siRNA
• Conservation
miRNA vs siRNA
miRNA in plants
• Near-perfect complementarity
• mRNA cleavage, usually of TF’s related to
developmental processes
• Conservation between Arabidopsis and
rice
• Defend against viruses
miRNA in animals
• mRNA cleavage or translational silencing
• Conservation is also high (?)
• Different numbers of paralogs
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
Goal
Find new human
micro RNAs
Motivation
Current gene search techniques:
• Hairpins
• Conservation
Try to search with a
wider scope
Search algorithm
Prediction (1)
Fold the genome
~ 11 milion
hairpins
Magic box
~ 430,000 hairpins
Magic box
Structure features
Sequence features
•Hairpin length
•Loop length
•Stability score
•Free energy per
nucleotide
•Matching pairs
•Bulge size
•Sequence repetitiveness
•Regular internal repeat
•Inverted internal repeat
Build a classifier
Prediction (2)
~ 430,000 hairpins
Non- conserved
sample
800
clustered
3000
nonclustered
conserved
sample
1500
clustered
7500
control
Prediction (3)
800
clustered
3000
nonclustered
1500
clustered
7500
control
Micro array in five tissue cultures
886 confirmed miRNA
Sample
359 miRNA
Validate (clone and sequence)
69 ‘adjacent’
miRNA
Prediction (3)
359 miRNA
69 ‘adjacent’
miRNA
Validate (clone and sequence)
89 (33 ‘adjacent’) cloned and
sequenced NEW miRNA
Of these:
•1 from the control list
•36 conserved miRNA’s (32 validated in other experiments)
•43 in two new clusters
New cluster
The cluster conatin a few ‘seeds’
Results summary
Goal
Location on
chromosome
Expression
?
Creating miRNA micro array
Design microRNA chip
• Normalization by synthetic samples
• Melting temperature
Array Results
Is Expression correlated with
distance between microRNA’s?
55bp
Is expression of micro RNA’s
correlated with host genes ?
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
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 ?