micro RNA’s

Computational biology seminar

Ariel Jaimovich

November 17th 2005

The central dogma of biology

• This is not always the case:– First ‘life forms’– viruses

Transcription

RNA

Translation

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 organism4 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 ?

Protein coding rna

3’ utr

Function (cont)• Plants vs animals

• Number of target seq. on 3’ utr ?

• Some miRNA target the same mRNA in different sites

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

Fold the genome

~ 11 milion hairpins

Prediction (1)

Magic box

~ 430,000 hairpins

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

Magic box

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

Build a classifier

Prediction (2)

~ 430,000 hairpins

conservedNon- conserved

samplesample

800 clustered

3000 non-

clustered

1500 clustered

7500control

Prediction (3)800

clustered 3000 non-

clustered

1500 clustered

7500control

Micro array in five tissue cultures

886 confirmed miRNA

Sample359 miRNA

Validate (clone and sequence)

69 ‘adjacent’ miRNA

Prediction (3)

359 miRNA

Validate (clone and sequence)

69 ‘adjacent’ miRNA

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

ExpressionLocation on chromosome

?

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 ?