Nobel price 2006 Andrew Z. Fire Craig C. Mello "for their discovery of RNA interference - gene silencing by double-stranded RNA" Andrew Z. Fire Craig C. Mello USA Stanford University School of Medicine Stanford, CA, USA University of Massachusetts Medical School Worcester, MA, USA

Serendipity? Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Andrew Fire, SiQun Xu, Mary K. Montgomery,Steven A. Kostas, Samuel E. Driver & Craig C. Mello NATURE | VOL 391 | 19 FEBRUARY 1998 They worked on something else in C. elegans Gene expression – sense RNA (control) Gene silencing – antisense RNA (weak effect) One more controla with double strand (s-as) RNA, SURPRISE – IT WORKED VERY WELL

Nobelova price experiment dsRNA blocks target mRNA asRNS dsRNS

Not absolute new Drosophila (Elbashir, EMBO J, 2001, Genes and Dev, 2001) dsRNA → 21-23 nt small dsRNA small interferring RNA – siRNA siRNS 21-25 nt dsRNA from a bigger dsRNA precursor molecule

These are coded in genome – also in ours C. elegans (Lee, 1993, Cell) NON CODING SEQUENCES! SMALL (61 → 22nt) RNA lin-14 mRNS 3’ UTR komplementerita REGULATION OF GENE EXPRESSION microRNA (miRNA)

miRNS biogenesis The code is transcribed by RNA polymerase II More possibilities – separated - 1 miRNA – clusters – in INTRONS! Mendell, 2008, Cell

Biogenesis and effect of siRNA Dykxhoorn, 2003, Nat Rev Mol Cell Biol

Finalisation of siRNA With protein complexes (coded in genome) Dicer makes siRNA (+ATP) RISC – komplex with siRNA, cuts mRNA (+ATP) Argonauta – blocks translation

siRNA effect Eckstein, 2005, TIBS

Another possibility Inhibícia translácie

miRNA - functions In human genome at least 500 – 1000 miRNa genes 1-1,5 % of all structural genes A new (?) form of gene expression regulation – on the interface of transcription and translation THE LAST CHECKPOINT (SMALL NINJA POLICIMEN) miRNA expression regulation???? tissue specificity, dependent on ontogenesis Involved in Development, differentiation, apoptosis, cell cycle

miRNA functions One miRNA can regulate more mRNA (Lim, 2005, Nature) In different conditions different picture of expression miR-1 – heart, miR-124 – brain In vitro experiments miR-1 96, miR124 downregulated 174 génov

miRNA and disease miRNA profiles different in normal and malignant cells miRNA profiles in diabetes, cardiovascular and kidney diseases, degenerative disease of CNS – intensive research currently without aplication in practical medicine SNPs in miRNA genes and disease??? Therapeutical perspectives? Malignancies Farmakogenetics Design of artifical siRNA? Not a simple task Get them to the proper place – nanobiotechnology

Fewell, 2006, Drug Discovery