Transcript 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 ?