RNA interference.

Presentazione sul tema: "RNA interference."— Transcript della presentazione:

1 RNA interference

2 phenotype RNA interference Forward genetics Reverse genetics
Ricombinazione omologa/genome editing in cellule embrionali staminali di topo: topi KO RNA antisenso RNA interference

3 Discovery of RNA interference: Silencing of target gene
flies fungi worms mammals plants Post-Transcriptional Gene Silencing (PTGS): Piante: transgeni ad alto numero di copie e altamente trascritti inducono il silenziamento del gene endogeno Può essere considerato come un primitivo sistema di autodifesa contro RNA esogeno (virus) o RNA endogeno trascritto in modo aberrante (trasposoni)

4 La Scoperta dell’ RNAi Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Fire et al, 1998 In C. elegans: introduzione di dsRNA molto più potente nell’indurre il silenziamento genico rispetto a sRNA e asRNA dsRNA corrispondenti a regioni introniche o al promotore non funzionano dsRNA microiniettato nelle gonadi induce silenziamento genico in tessuti: amplification/spreading Targeted mRNA degradation by double stranded RNA in vitro Tuschl et al, 1999 In Drosophila: In vitro: un estratto citoplasmatico di Drosophila + luciferasi mRNA esogeno + dsRNA luciferasi: silenziamento genico dsRNA guida la formazione di un complesso citoplasmatico che determina la degradazione specifica di mRNA omologo RNA interfernce is mediated by 21 and 22-nucleotide RNAs Elbashir et al, 2001 In Drosophila: dsRNA è processato a nt dsRNA con 5’P, 2nt protruding 21-22-dsRNA sono sufficienti a indurre RNAi

5 Mechanism of RNAi DICER: RNAse III: Ribonuclease specific fordsRNA
Highly conserved in flies, plants, worms e human RISC-RNA Induced Silencing Complex SLICER Complex with ARGONAUTE EIFC2 (elongation factor) and Helicase DICER RISC Production of siRNA 21-22 dsRNA, 5’P, 2nt overhang Riconoscimento e taglio endonucleasico del mRNA target

6 Inattivazione dell’RNAi causa diffetti nello sviluppo
in diversi organismi Mutationi in ARGONAUTE (dsRNA-binder in RISC) causano difetti nello sviluppo in A. thaliana, C. elegans e Drosophila microRNA piccoli (21-22nt) ssRNA scoperti in C. elegans (Lin4 and Let7: worms development) Altamente conservati nell’evoluzione Silenziamento genico a livello traduzionale: mRNA binding-blocco sintesi proteica 21-22 nt single strand RNA processati da dsRNA precursori a 70nt (miRNA). Link to RNAi: DICER ko hanno alti livelli miRNA precursori di Lin4 e Let7 RISC DICER

7 miRNA in human

8 Antisense RNAs in humans

9 The non coding genome mRNA ncRNA GENE PROTEINA miRNA asRNA tRNA rRNA
snRNA: spliceosome snoRNA: rRNA-tRNA-snRNA modifications rRNA GENE PROTEINA Cis-AS-RNA: XIST miRNA asRNA

10 RNAi un nuovo strumento per lo studio della funzione genica nei mammiferi
Limitation long dsRNA >30nt is effective in mammals, but induce protective pathways Long dsRNA trigger PKR IFN RNAseL apoptosis dsRNA degradation Block of protein synthesis Phosp of ElF2a

11 Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature (2001). 2nt increase stability easier synthesis Source for siRNA: Dharmacon, MWG, Ambion. When sequence known, order to any oligo-producing Company.

12 Design the perfect RNAi
1) stability of the hairpin 2) access to RISC 9nt loop 2nt TT overhang Low GC content Empirical observation Structure of miRNA

13 Rules for siRNA design Mittal, Nature Review Gentic, 2004 29 su 30 dsRNA inducono più 50% silencing su 6 geni target

14 siRNA Vettori a DNA per RNAi ? CONTRO PRO TRANSIENT EFFECT
Rate of cell growth? Dilution effect: short time inactivation Ineffectual with Proteis with long half-life EFFICIENCY OF TRANSFECTION Some cells are refractory to transfection Electroporation leads to cell death COST siRNA are chemically sinthetized, therefore expensive NOT RENEWABLE Use in vivo????? siRNA transfection is very efficient in most cell lines siRNA silencing is immediate Vettori a DNA per RNAi ?

15 Plasmid vector

16 2) shRNA vectors. PRO CONTRO Promotori RNA Pol III: U6 e H1
Syntesis of short hairpin RNA (shRNA) in vivo PRO Cheap (They ca be propagated) Stable clones for long-term experiments Proteins with long half life targeted CONTRO Interference requires transcription: timing! Transfection is less efficient More efficiently processed by DICER! Plasmid vectors VIRUS vector mediated RNAi: H1/U6 in retroviral: stable integration in the genome oncoretrovirus based on MoMuLV or MSCV silencing of provirus during development cells must be cycling! lentivirus based on HIV-1 infections of quiescent cells

17 Check Specificity! NO YES
siRNA against endogenous gene can affect other genes with weak sequence similarity siRNA against endogenous GFP expression didn’t affecct any other gene Use indipendent siRNA against same target Rescue the observed Loss of Funtion phenotype by expressing the target gene with a silent mutation in the target region

18 2) Limitations of stable RNAi :
impossible to study genes essential for cell survival (housekeeping) or development Vectors for conditional-inducible expression of shRNA Tet-OFF/ON H1 and U6 promoter system toxicity background

19 Loss of funtion screenings by RNAi

20 ? Sequenziamento del genoma umano
Annotazione di tutte le possibili ORF Costruzione di siRNA contro tutti i geni umani ? Genome wide functional screenings

21 with siRNA/shRNA library
Infect cells with siRNA/shRNA library Phenotype of interest The targeted gene is required for the phenotype How can we identify the targeted gene? PCR with primer flanking the siRNA encoding DNA sequnece cloning sequencing

22 functional screenings
High-throughput functional screenings

23 Limitations: single clone isolation re-cloning and retesting
Infect cells select phenotype Single cell derived clones Re-Cloning and sequencing Test again siRNA BAR-CODE screens Stably integraetd vectors introduce a molecular fingerprint in the infected cell: The 19 hairpin sequence is gene specific and behave as a MOLECULAR BARCODE Relative abundance of BARCODE in a cell population reflect the effect of the knockdown on cell viability Infect cells select population with phenotype PCR of hairpin sequence Fluorescent labeling Hybridize to DNA Microarray containing hairpin sequences from library Identification of highly represented shRNA

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25 Silenziamento genico specifico, efficiente e, potenzialmente, stabile nel tempo.
Genetica inversa economica e veloce. Rivoluzione nella comprensione dei meccanismi di regolazione dell’espressione genica. Genome-wide functional screenings.