Dogmatic View of Gene Expression DNA Protein RNA 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)
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Dogmatic View of Gene Expression DNAProteinRNA Post-transcriptional Control: Quantitative Control: Levels of mRNA not proportional to levels of mRNA synthesized.
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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 ofGene Expression
RNADNA Protein
RNA: A Diverse Class of Molecules
RNADNA
Non Coding RNAs: ‘RiboRegulators’
(~97% of RNAs Present in Human Cells are Non-Coding)
rRNAtRNA
snRNAssnoRNAsGuide RNA
Catalytic:RibozymesTelomerase
Introns5’ UTR3’ UTR
VaultY RNAs7SK
Xist, H19
MicroRNAs
Viral RNAsRetrotransposons
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
Double-stranded RNA
inject
C. elegans
Neg. control Uninjected
Antisense RNA dsRNA
sense
antisense
Nature 1998 391:806-811
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
~22nt siRNAs
targetmRNA
secondary siRNAs
amplification
processing
degradation
recognition
copying+
processing
spreading
Gene Silencing Factors
C. elegansDrosophila Arabidopsis
amplification
processing
degradation
recognition
copying+
processing
spreadingDICERDCR-1CAF
AGO2RDE-1AGO1R
ISC
SID-1RRF-1SDE-1/SGS-2
RDE-4
VIGCG1800
Fmr1
RNAi in Mammalian Cells• Long dsRNA triggers global (non-specific) gene-silencing (i.e. interferon response)• Breakthrough: Short dsRNA (~22 nt) induces RNAi
Nature 2001 411: 494-498
Silencing of lamin proteins in human cells by dsRNA transfection
RNA-Mediated Gene Silencing
RNA Interference‘Cosuppression’ by transgenes in plants
‘Quelling’ in FungiTranscriptional Gene Silencing (TGS)
Common Trigger:
RNAi Applications
GENETIC TOOL
GENE THERAPY
Probing Gene Function
Combat Viral InfectionTreat Genetic Diseases
(New expression strategies)
What is the Purpose of RNA-Mediated
Gene Silencing?
Proposed Biologic Roles
• Antiviral Defense• Suppress Transposon Activity• Response to Aberrant RNAs
• Gene Regulation (e.g. MicroRNAs)
‘Immune System’ of the Genome
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 Developmental regulationby stRNAs (µ RNAs)