Download complesso EJC Non-sense Mediated Decay (NMD)

Non-sense Mediated Decay (NMD):
complesso EJC
Non-sense Mediated Decay (NMD)
"Pioneer round"
of translation
Meccanismo dell’NMD
AUG
UAG (PTC)
mGppp
3
22
mGppp
STOP
3
3
3
AAAAAAAAAAAAAAAA
1 2
3
RF
AAAAAAAAAAAAAAAA
UAG
2
mGpp
3
p
UAG
AAAAAAAAAAAAAAAA
Degradazione normale
Degradazione NMD
Non-Stop Mediated Decay (NSMD)
RNA interference
• Meccanismo
• Significato biologico
• Strumento di analisi della funzione dei geni
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
Componenti dell’RNAi
• 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)
Inizio dell’RNAi
• dsRNA (80-100 nt) in piante, C. elegans, Drosophila
• siRNA in mammiferi
Effetto del dsRNA
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.
Meccanismo
1. Produzione siRNA
2. Formazione complesso RISC
3. Attivazione complesso RISC
4. Taglio dell’RNA bersaglio
Propagazione dell’RNAi
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.
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 RNAdependent 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.
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 RNAdirected RNA polymerase; blue and red RNA, doublestranded 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-IIIlike dsRNA-specific ribonuclease; RdRP, an RNAdirected 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].
Caratteristiche dell’RNAi
• Può agire a diversi livelli: trascrizionale, posttrascrizionale, traduzionale
• Può amplificarsi e diffondersi nell’organismo
• Può coinvolgere sequenze adiacenti
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.
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 dell’espressione genica
• Mutazioni in componenti dell’RNAi causano alterazioni dello
sviluppo (Arabidopsis, C. elegans, Drosophila)
• Regolazione del gene Stellate in Drosophila
Ruolo biologico dell’RNAi
• Difesa dai virus (nelle piante VISG)
• Controllo dei trasposoni
• Regolazione dell’espressione genica
Uso dell’RNAi
• Studio sistematico della funzione dei geni
• Terapia genica
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.
Small temporal RNA (stRNA)
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
Strutture secondarie dei pre-miRNA
Organizzazione dei geni di miRNA
Biogenesi dei miRNA
Biogenesi dei miRNA
Kosik Nature Reviews Neuroscience 7, 911–920 (December 2006) | doi:10.1038/nrn2037
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 RNAdirected 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.
Kosik Nature Reviews Neuroscience 7, 911–920 (December 2006) | doi:10.1038/nrn2037
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.
CARATTERISTICHE DI UN "SISTEMA VIVENTE"
replicazione
evoluzione
DOGMA CENTRALE
DNA
RNA
proteine
CAPACITA' DIVERSE DELL'RNA
RNA come genoma: virus a singolo o doppio
filamento
RNA come catalizzatore: meccanismo basato
su cationi bivalenti (metalloribozimi)
Apparecchio
di Miller (1953)
DIFFICOLTA' DELLE SINTESI PREBIOTICHE
rese basse
ribosio instabile (isomeri)
legame glicosidico a o b
fosfato solo al 3' e 5'
legami fosfodiesterici solo 5'-3'
FUNZIONI DELL'RNA NELLA CELLULA MODERNA
traduzione
rRNA, mRNA, tRNA
maturazione rRNA
snoRNA, RNasi MRP
splicing
snRNA, introni gruppi I e II
maturazione tRNA
RNasi P
sintesi DNA
primers, telomerasi
traslocaz. proteine
srpRNA
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
EVOLUZIONE SINTESI PROTEICA
1) tRNA
2) tRNA sintetasi
3) protoribosoma
4) codice genetico
5) mRNA
6) proteine
PASSAGGIO RNA
DNA
1) sintesi dei precursori del DNA
2) trascrizione inversa
3) replicazione
4) trascrizione
SELEX
(SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT)
es. affinity column