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Post-transcriptional Control:

DNA. RNA. Protein. Post-transcriptional Control:. Dogmatic View of Gene Expression. 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

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Post-transcriptional Control:

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  1. DNA RNA Protein Post-transcriptional Control: Dogmatic View of Gene Expression 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)

  2. An “RNA-Centric” View of Gene Expression RNA DNA Protein

  3. RNA: A Diverse Class of Molecules RNA DNA

  4. Non Coding RNAs: ‘RiboRegulators’ (~97% of RNAs Present in Human Cells are Non-Coding) rRNA tRNA snRNAs snoRNAs Guide RNA Catalytic: Ribozymes Telomerase Vault Y RNAs 7SK Introns 5’ UTR 3’ UTR MicroRNAs Viral RNAs Retrotransposons Xist, H19

  5. Non Coding RNAs: • SnoRNAs • Large Family • Intron-encoded • Guide RNA Modification

  6. Non Coding RNAs: • Telomerase RNA • Component of telomerase • Provides template for • telomere synthesis • Role in Cancer and Aging

  7. 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

  8. Discovery of RNAi Double-stranded RNA Neg. control Uninjected sense antisense inject Antisense RNA dsRNA Mex-3 mRNA detection in embryos by in situ hybridization C. elegans Nature 1998 391:806-811

  9. 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)

  10. Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs dsRNA processing ~22nt siRNAs recognition amplification target mRNA spreading copying degradation + processing secondary siRNAs

  11. DICER DCR-1 CAF processing AGO2 RDE-1 AGO1 recognition RISC amplification spreading copying degradation + processing VIG CG1800 Arabidopsis RRF-1 SDE-1/SGS-2 RDE-4 SID-1 Drosophila C. elegans Fmr1 Gene Silencing Factors

  12. 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

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

  14. RNAi Applications GENETIC TOOL Probing Gene Function GENE THERAPY Combat Viral Infection Treat Genetic Diseases (New expression strategies)

  15. What is the Purpose of RNA-Mediated Gene Silencing?

  16. Proposed Biologic Roles ‘Immune System’ of the Genome • Antiviral Defense • Suppress Transposon Activity • Response to Aberrant RNAs • Gene Regulation (e.g. MicroRNAs)

  17. RNA-Mediated Gene Silencing Science 2002 296:1263-1265

  18. 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!

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

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