Download RNA-Mediated Gene Silencing

Dogmatic View of
Gene Expression
DNA
RNA
Protein
Post-transcriptional Control:
Quantitative Control:
Levels of mRNA not proportional to levels of
mRNA synthesized or protein produced
Qualitative Control:
More than one protein from a single gene
(e.g. Differential RNA Processing or RNA editing)
An “RNA-Centric” View of
Gene Expression
DNA
RNA
Protein
RNA: A Diverse Class of Molecules
DNA
RNA
Non Coding RNAs: ‘RiboRegulators’
(~97% of RNAs Present in Human Cells are Non-Coding)
rRNA
tRNA
Vault
Y RNAs
7SK
Xist, H19
snRNAs
snoRNAs
Guide RNA
Introns
5’ UTR
3’ UTR
Catalytic:
Ribozymes
Telomerase
MicroRNAs
Viral RNAs
Retrotransposons
Non Coding RNAs:
SnoRNAs
• Large Family
• Intron-encoded
• Guide RNA Modification
Non Coding RNAs:
Telomerase RNA
• Component of telomerase
• Provides template for
telomere synthesis
• Role in Cancer and Aging
RNA-Mediated Gene Silencing
Post-transcriptional Gene Silencing (PTGS)
or RNA Interference (RNAi)
Transcriptional Gene Silencing (TGS)
(RNA-dependent DNA Methylation)
Gene Silencing By MicroRNAs
Discovery of RNAi
Nature 1998 391:806-811
Double-stranded RNA
sense
Neg. control
Uninjected
antisense
inject
C. elegans
Antisense RNA
dsRNA
Mex-3 mRNA detection in embryos
by in situ hybridization
Remarkable Properties of RNAi
• dsRNA (not ssRNA) is interfering agent
• Sequence-specific loss of mRNA and protein
• Effective against exons not introns
• Potent (few dsRNA molecules/cell effective)
• Persistent (affects next generation)
• Effects can cross cell barriers (feed, soak)
Mechanism of RNAi: Gene Silencing
directed by ~22nt RNAs
dsRNA
processing
~22nt siRNAs
amplification
target
mRNA
degradation
spreading
recognition
copying
+
processing
secondary
siRNAs
Gene Silencing Factors
DICER
DCR-1
CAF
processing
AGO2
VIG recognition
RDE-1 CG1800
AGO1 Fmr1
degradation
Drosophila
amplification
RRF-1
SDE-1/SGS-2
spreading
RISC
RDE-4
SID-1
copying
+
processing
C. elegans
Arabidopsis
RNAi in Mammalian Cells
• Long dsRNA triggers global (non-specific)
gene-silencing (i.e. interferon response)
• Breakthrough: Short dsRNA (~22 nt) induces RNAi
Silencing of lamin proteins in human cells by dsRNA transfection
Nature 2001 411: 494-498
RNA-Mediated Gene Silencing
RNA Interference
‘Cosuppression’ by transgenes in plants
‘Quelling’ in Fungi
Transcriptional Gene Silencing (TGS)
Common Trigger:
RNAi Applications
GENETIC TOOL
Probing Gene Function
GENE THERAPY
Combat Viral Infection
Treat Genetic Diseases
(New expression strategies)
What is the Purpose
of RNA-Mediated
Gene Silencing?
Proposed Biologic Roles
‘Immune System’ of the Genome
• Antiviral Defense
• Suppress Transposon Activity
• Response to Aberrant RNAs
• Gene Regulation (e.g. MicroRNAs)
RNA-Mediated Gene Silencing
Science 2002 296:1263-1265
MicroRNAs:
Expanding Family of ‘RiboRegulators’
•
•
•
•
lin-4 and let-7 RNAs (from worm) were first examples
Also known as stRNAs (small temporal RNAs)
Regulate expression of proteins and developmental timing
Tip of the iceberg………..MicroRNAs are everywhere!
RNAi by siRNAs
~22nt
siRNAs
Developmental regulation
by stRNAs (µ RNAs)
processing
~22nt
lin-4
target
recognition
mRNA
lin-14
mRNA
lin-41
mRNA
processing
~22nt
let-7
target
recognition
3’UTR
3’UTR
degradation
Translational repression