RNA interference (RNAi) and its applica3ons. Carlos Camilleri

Transcription

1 RNA interference (RNAi) and its applica3ons Carlos Camilleri

2 Content of the presenta3on Introduc1on Argonaute proteins pirnas: biogenesis and gene silencing mirna and sirna Differences Biogenesis Gene Silencing Transla1onal ac1va1on Applica1ons of RNA interference Poten1al therapeu1c applica1on in gene1c diseases Poten1al an1viral response: HIV Current drawbacks of using RNAi Conclusion

3 Introduc3on RNA interference is a process of gene silencing Three different types of RNA involved: Micro interference RNA (mirna) Small interference RNA (sirna) Piwi- interac1ng RNA (pirna) RNA- induced silencing complex (RISC)

4 Argonaute family Highly specialized small- RNA- binding proteins Key of RNA- silencing pathways 2 human subfamilies with 4 members each: Subfamily Expression Associated to AGO Ubiquitous mirnas and sirnas PIWI Germline pirnas 10- fold increase in finding target mrna

5 pirnas biogenesis and gene silencing Derive from transposons à share homology Transcribed from pirna clusters: Long single- stranded transcript Sense or an1sense transcrip1on Biogenesis of pirnas follows 2 different pathways: Primary processing pathway Amplifica1on pathway (ping- pong cycle) Mature pirnas are nt in length Mechanism of transposon silencing in mammals

6 pirnas biogenesis and gene silencing pirna cluster Primary pirnas Zuc Primary processing Sense or antisense transcript Antisense transcript PIWI protein Sense pirna Secondary processing (ping-pong cycle) Antisense pirna Mature sense pirna Trimming Sense transcript Trimming Mature antisense pirna

7 mirna and sirna O[en referred to as the true interference RNA Similar biogenesis but s1ll key differences Mature structure TargeBng mirna dsrna of 19-25nt with a 2nt 3'overhang Mul1ple targets, par1ally complementary sirna dsrna of 21-23nt with a 2nt 3'overhang One highly specific target with fully complementariety Mechanisms of gene regula3on 1. Transla1onal repression 2. mrna degrada1on 3. Endonucleotydic cleavage 1. Endonucleotydic cleavage

8 Biogenesis of mirna and sirna Both form RNA duplexes (dsrna) a[er transcrip1on mirna requires an extra processing in the nucleus Both are exported to the cytoplasm by Expor1n- 5 In the cytoplasm, their processing is the same

9 Drosha mirna gene Transcription Pri-miRNA Pre-miRNA Pol II Exportin 5 Nucleus dsrna Cytoplasm sirna DICER mirna RISC RISC AGO AGO Passenger strand is cleaved Activated RISC mirisc Passenger strand is discarded Complementary binding of the guide strand to target mrna Incomplete complementary binding of the guide strand to target mrna mrna mrna 3 UTR mrna cleavage Translational repression, mrna degradation, mrna cleavage

10 mirna and sirna gene silencing Endonucleotydic cleavage of target mrna Performed by the AGO2 Needs perfect complementarity Cleavage followed by mrna degrada1on

11 V413-BI79-13 ARI mirna- only gene silencing 27 April :36 Transla3onal repression a Initiation block Repressed Cap recognition Repressed 60S subunit joining 40S mirisc GW182 AGO m7g eif4e AUG 60S Open reading frame PABP A(n)

12 Repressed 60S subunit joining 40S mirisc mirna- only gene silencing m7g AUG GW182 AGO PABP A(n) Open reading frame Transla3onal 60Srepression eif4e b Postinitiation block Elongation block Proteolysis 40S mirisc Nascent polypeptide Ribosome drop-off GW182 AGO eif4e Figure 2 m7g PABP A(n)

13 mirna- only gene silencing mrna degrada3on AUG 2 Decapping (and subsequent decay) m 7 G DCP1/ DCP2 Open reading frame CAF1 CCR4 NOT1 AGO mirisc 1 Deadenylation GW182 PABP A (n) Figure 3 Schematic diagram of mirna-mediated mrna decay. The mirisc interacts with the CCR4-NOT1 deadenylase complex to facilitate deadenylation of the poly(a) tail [denoted by A (n) ]. Deadenylation requires

14 Transla3onal ac3va3on by mirna Mechanism s1ll under study Only found in quiescent cells arrested in G0/G1 Involves recruitment of FXR1 (fragile X- related protein 1)

15 Applica3ons of RNA interference Poten1al therapeu1cs for gene1c diseases Dominant gene1c disorders caused by a mutant allele in the presence of a second, normal allele Currently no cures: difficult to treat the origin, in some cases the muta1on is a SNP Demonstrated in vitro that a SNP is enough for RNAi to select the mutant allele over the normal one S1ll under development

16 Applica3ons of RNA interference Poten1al an1viral responses: HIV Successful targe1ng of HIV- encoded RNAs Successful downregula1on of cellular cofactors needed for the HIV replica1on Inhibi1on of HIV replica1on achieved in several human cell lines Big challenge going from in vitro to in vivo High viral muta1on rate à escaping mutants Challenging delivery of the RNAi to the infected cells S1ll under development

17 Current major drawbacks of using irna Off- target effects Gene silencing of unwanted genes Delivery methods Large size of irnas (around 14 kda) Nega1ve charge of RNA Generally unstable in vivo Problems targe1ng specific cells or 1ssues

18 Conclusion Clinical trials with RNAi are currently underway, but major obstacles, such as off- target effects, toxicity and unsafe delivery methods, have to be overcome before RNAi can be considered a conven1onal drug

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