Computational biology seminar micro RNA’s Computational biology seminar Ariel Jaimovich November 17th 2005

The central dogma of biology Transcription RNA Translation Protein This is not always the case: First ‘life forms’ viruses

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 Protein coding rna 3’ utr

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

Find new human micro RNAs 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

Magic box Structure features Sequence features Build a classifier Hairpin length Loop length Stability score Free energy per nucleotide Matching pairs Bulge size Sequence features Sequence repetitiveness Regular internal repeat Inverted internal repeat Build a classifier

Prediction (2) ~ 430,000 hairpins conserved Non- conserved sample 800 clustered 3000 non-clustered 1500 clustered 7500 control sample

Micro array in five tissue cultures Validate (clone and sequence) Prediction (3) 800 clustered 3000 non-clustered 1500 clustered 7500 control Micro array in five tissue cultures 886 confirmed miRNA 69 ‘adjacent’ miRNA Sample 359 miRNA Validate (clone and sequence)

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 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 Conclusions 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 ?