Dogmatic View of Gene Expression DNAProteinRNA 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 RNA DNA Protein

RNA: A Diverse Class of Molecules RNA DNA

Non Coding RNAs: RiboRegulators (~97% of RNAs Present in Human Cells are Non-Coding) rRNA tRNA snRNAs snoRNAs Guide RNA Catalytic: Ribozymes Telomerase Introns 5 UTR 3 UTR Vault Y RNAs 7SK Xist, H19 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 Double-stranded RNA inject C. elegans Neg. control Uninjected Antisense RNA dsRNA sense antisense Nature : 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 target mRNA secondary siRNAs amplification processing degradation recognition copying + processing spreading

Gene Silencing Factors C. elegans Drosophila Arabidopsis amplification processing degradation recognition copying + processing spreading DICER DCR-1 CAF AGO2 RDE-1 AGO1 RISC SID-1 RRF-1 SDE-1/SGS-2 RDE-4 VIG CG1800 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 : Silencing of lamin proteins in human cells by dsRNA transfection

RNA-Mediated Gene Silencing RNA Interference Cosuppression by transgenes in plants Quelling in Fungi Transcriptional Gene Silencing (TGS) Common Trigger:

RNAi Applications GENETIC TOOL GENE THERAPY Probing Gene Function Combat Viral Infection Treat 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 :

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 regulation by stRNAs (µ RNAs) processing ~22nt siRNAs ~22nt lin-4 processing target recognition target recognition mRNA lin-14 mRNA lin-41 mRNA 3UTR degradation ~22nt let-7 Translational repression