Non-sense Mediated Decay (NMD): complesso EJC. Non-sense Mediated Decay (NMD)

Presentazione sul tema: "Non-sense Mediated Decay (NMD): complesso EJC. Non-sense Mediated Decay (NMD)"— Transcript della presentazione:

1 Non-sense Mediated Decay (NMD): complesso EJC

2 Non-sense Mediated Decay (NMD)

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4 "Pioneer round" of translation

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6 3 2 Meccanismo dellNMD UAG (PTC)AUGSTOP mGppp AAAAAAAAAAAAAAAA 33 2 mGpp p AAAAAAAAAAAAAAAA 3 UAG AAAAAAAAAAAAAAAA mGppp UAG 33 22 1 RF

7 Degradazione normale

8 Degradazione NMD

9 Non-Stop Mediated Decay (NSMD)

10 RNA interference Meccanismo Significato biologico Strumento di analisi della funzione dei geni

11 Co-soppressione Introduzione di copie transgeniche di un gene risulta nella ridotta espressione del transgene e del gene endogeno RNA interference Introduzione di RNA a doppio filamento (dsRNA) induce silenziamento genico

12 Componenti dellRNAi siRNA (small interfering RNA) = frammenti di RNA 21-25 nt Dicer = endoribonucleasi specifica per dsRNA (tipo RNasi III) RISC (RNA-induced silencing complex) = complesso ribonucleoproteico RdRP (RNA-dependent RNA polymerase)

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14 Inizio dellRNAi dsRNA (80-100 nt) in piante, C. elegans, Drosophila siRNA in mammiferi

15 Effetto del dsRNA

16 Figure 2 Dicer and RISC (RNA-induced silencing complex). a, RNAi is initiated by the Dicer enzyme (two Dicer molecules with five domains each are shown), which processes double-stranded RNA into ~22-nucleotide small interfering RNAs. Based upon the known mechanisms for the RNase III family of enzymes, Dicer is thought to work as a dimeric enzyme. Cleavage into precisely sized fragments is determined by the fact that one of the active sites in each Dicer protein is defective (indicated by an asterisk), shifting the periodicity of cleavage from ~9–11 nucleotides for bacterial RNase III to ~22 nucleotides for Dicer family members. The siRNAs are incorporated into a multicomponent nuclease, RISC (green). Recent reports suggest that RISC must be activated from a latent form, containing a double-stranded siRNA to an active form, RISC*, by unwinding of siRNAs. RISC* then uses the unwound siRNA as a guide to substrate selection31. b, Diagrammatic representation of Dicer binding and cleaving dsRNA (for clarity, not all the Dicer domains are shown, and the two separate Dicer molecules are coloured differently). Deviations from the consensus RNase III active site in the second RNase III domain inactivate the central catalytic sites, resulting in cleavage at 22-nucleotide intervals.

17 Meccanismo 1.Produzione siRNA 2.Formazione complesso RISC 3.Attivazione complesso RISC 4.Taglio dellRNA bersaglio

18 Propagazione dellRNAi

19 Figure 3 Transitive RNAi. In transitive RNAi in C. elegans, silencing can travel in a 3 to 5 direction on a specific mRNA target. The simplest demonstration comes from the creation of fusion transcripts. Consider a fragment of green fluorescent protein (GFP) fused to a segment of UNC-22 (left). Targeting GFP abolishes fluorescence but also creates an unexpected, uncoordinated phenotype. This occurs because of the production of double-stranded RNA and consequently small interfering RNAs homologous to the endogenous UNC-22 gene. In a case in which GFP is fused 5 to the UNC-22 fragment (right), GFP dsRNA still ablates fluorescence but does not produce an uncoordinated phenotype.

20 An integrated model for RNAi and PTGS. In this model, the sequential action of Dicer (to generate siRNAs) and Slicer (to cleave the target RNA) are considered the primary route for target destruction. Amplification of the siRNAs is postulated to occur by either (or both) random degradative PCR or production of siRNAs from aberrant RNA that is, the copying of the target RNA or a cleavage product of the target RNA by an RNA- dependent RNA polymerase to generate a dsRNA substrate for Dicer, thereby creating new siRNAs. In the random degradative PCR scheme, the polymerase is envisioned to be primed by an siRNA guide strand. Conversion of aberrant RNA to dsRNA is drawn here unprimed.

21 Fig. 1. Models of molecular pathways involved in double-stranded RNA (dsRNA)-mediated silencing. The basic mechanism, which is probably present in most eukaryotes undergoing dsRNA-mediated silencing, is indicated by the gray and blue boxes. The remaining steps seem to occur in at least some organisms, but their generality is currently unknown and their subcellular location is mainly hypothetical. The triggers of silencing, either direct sources of dsRNA or transcription units producing singlestranded RNAs that can be presumably converted to dsRNA, are colored red. Green RNA, endogenously transcribed single-stranded RNA; purple RNA, RNA synthesized by a putative RNA- directed RNA polymerase; blue and red RNA, double- stranded RNA introduced exogenously or resulting from viral replication, annealing of complementary ssRNAs and/or hairpin RNA. Proteins or protein complexes are indicated by yellow boxes: CAF, an Arabidopsis homolog of Dicer; Dicer, an RNase-III- like dsRNA-specific ribonuclease; RdRP, an RNA- directed RNA polymerase; and RISC, RNA-induced silencing complex. Although dsRNA is depicted in single nuclear and cytoplasmic pools, depending on the source these molecules might be delivered differently to the processing Dicer enzymes. Similarly, the RISC and RISC-like complexes might have different components and associated effector proteins depending on their functions. Although a role for dsRNA in directing methylation of homologous DNA sequences has been demonstrated in plants, the molecular machinery involved in this process and the actual nature of the guide RNA have not been resolved. Recent evidence suggests that the RISC complex is equivalent to the miRNP complex (Fig. 2) in human cells [17].

22 Caratteristiche dellRNAi Può agire a diversi livelli: trascrizionale, post- trascrizionale, traduzionale Può amplificarsi e diffondersi nellorganismo Può coinvolgere sequenze adiacenti

23 Viral Suppressors of RNAi The genomes of three positive-strand RNA viruses that encode suppressors of RNAi (black boxes) are shown. The polymerase genes (white boxes), other viral proteins (gray boxes), viral protease cleavage sites (diamonds), and sgRNA promoters (bent arrows) are indicated for comparison.

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25 Controllo di acidi nucleici parassiti C. elegans senza RNAi hanno una aumentata mobilità di trasposoni endogeni In alcuni sistemi i trasposoni silenziati si trovano organizzati in eterocromatina Regolazione dellespressione genica Mutazioni in componenti dellRNAi causano alterazioni dello sviluppo (Arabidopsis, C. elegans, Drosophila) Regolazione del gene Stellate in Drosophila

26 Ruolo biologico dellRNAi Difesa dai virus (nelle piante VISG) Controllo dei trasposoni Regolazione dellespressione genica

27 Uso dellRNAi Studio sistematico della funzione dei geni Terapia genica

28 Fig. 1. The DNA vector-based RNA interference (RNAi) technology. (a) Generation of a hairpin siRNA directed by a Pol III promoter. An inverted repeat is inserted at the ©1 position of the U6 promoter (2351 to ©1). The individual motif is 19–29 nt, corresponding to the coding region of the gene of interest. The two motifs that form the inverted repeat are separated by a spacer of three to nine nt. The transcriptional termination signal of five Ts are added at the 30 end of the inverted repeat. The resulting RNA is predicted to fold back to form a hairpin dsRNA as shown. The resulting siRNA starts with either a G or an A at the 50 end, dependent on the promoter used (U6 or H1) and ends with one to four uridines, forming a 30 overhang that is not complementary to the target sequences. (b) Generation of two complementary siRNA strands synthesized by two U6 promoters. Two U6 promoters either placed in tandem or on two separate plasmids (not shown) direct transcription of a sense and an antisense strand of 19-nt RNAs. The two RNA strands are predicted to form a duplex siRNA in the transfected cells, with 30 overhangs of one to four uridines.

29 Small temporal RNA (stRNA)

30 Micro RNA (miRNA) Piccole molecole di RNA (20-22 nt) Prodotte da precursori di 90-100 nt trascritti autonomamente (anche policistronici) o maturati da introni Si legano a mRNA che hanno sequenze complementari Presenti in tutti gli eucarioti

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32 Strutture secondarie dei pre-miRNA

33 Organizzazione dei geni di miRNA

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35 Biogenesi dei miRNA

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37 Kosik Nature Reviews Neuroscience 7, 911–920 (December 2006) | doi:10.1038/nrn2037

38 Figure 5 A model for the mechanism of RNAi. Silencing triggers in the form of doublestranded RNA may be presented in the cell as synthetic RNAs, replicating viruses or may be transcribed from nuclear genes. These are recognized and processed into small interfering RNAs by Dicer. The duplex siRNAs are passed to RISC (RNA-induced silencing complex), and the complex becomes activated by unwinding of the duplex. Activated RISC complexes can regulate gene expression at many levels. Almost certainly, such complexes act by promoting RNA degradation and translational inhibition. However, similar complexes probably also target chromatin remodelling. Amplification of the silencing signal in plants may be accomplished by siRNAs priming RNA- directed RNA polymerase (RdRP)-dependent synthesis of new dsRNA. This could be accomplished by RISC-mediated delivery of an RdRP or by incorporation of the siRNA into a distinct, RdRP-containing complex.

39 Kosik Nature Reviews Neuroscience 7, 911–920 (December 2006) | doi:10.1038/nrn2037

40 Scheme for the function of different snmRNAs by targeting bacterial or eukaryal mRNAs or pre-mRNAs leading to regulation of gene expression. Binding of a snoRNA to the splice-site of a eukaryal pre-mRNA or involvement in mRNA editing and modification has recently been proposed as one of several alternatives for a functional role, but remains to be experimentally verified.

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43 CARATTERISTICHE DI UN "SISTEMA VIVENTE" replicazione evoluzione

44 DOGMA CENTRALE DNA RNA proteine

45 CAPACITA' DIVERSE DELL'RNA RNA come genoma: virus a singolo o doppio filamento RNA come catalizzatore: meccanismo basato su cationi bivalenti (metalloribozimi)

46 Apparecchio di Miller (1953)

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48 DIFFICOLTA' DELLE SINTESI PREBIOTICHE rese basse ribosio instabile (isomeri) legame glicosidico o fosfato solo al 3' e 5' legami fosfodiesterici solo 5'-3'

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53 FUNZIONI DELL'RNA NELLA CELLULA MODERNA traduzione rRNA, mRNA, tRNA maturazione rRNAsnoRNA, RNasi MRP splicing snRNA, introni gruppi I e II maturazione tRNARNasi P sintesi DNA primers, telomerasi traslocaz. proteine srpRNA

54 PERCHE' L'RNA? l'RNA deve essere venuto prima del DNA l'RNA deve essere venuto prima delle proteine molti coenzimi hanno un ribonucleotide nella struttura l'RNA può agire come catalizzatore

55 EVOLUZIONE SINTESI PROTEICA 1) tRNA 2) tRNA sintetasi 3) protoribosoma 4) codice genetico 5) mRNA 6) proteine

56 1) sintesi dei precursori del DNA 2) trascrizione inversa 3) replicazione 4) trascrizione PASSAGGIO RNA DNA

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60 SELEX (SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT) es. affinity column

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