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Judith Pagan Nov 2010

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Judith Pagan Nov 2010

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  1. The 2006 Nobel Prize in Physiology or Medicine was awarded to Andrew Fire and Craig Mello. It honours a discovery that has transformed biological research and may, in the future, prove useful in treating human disease. The discovery is called RNA interference, or RNAi.Describe the basic principles of how siRNA (small interfering RNA) and miRNA (microRNA) regulate gene expression. Describe the possible physiological roles of this process, how this discovery has provided critical biological reagents for functional genomics (give examples) and describe how it may be useful for therapy of certain diseases (give examples and describe possible risks of the methodology). Judith Pagan Nov 2010

  2. RNA interference (RNAi) • RNAi is a natural process that cells use to turn down, or silence the activity of specific genes • Post-transcriptional gene silencing • It is a highly conserved cellular defence mechanism • It directs development and gene expression • microRNA (miRNA) and small interfering RNA (siRNA) are central to RNAi • The miRNA and siRNA pathways are interchangeable • Small RNAs can bind to specific other RNAs eg. preventing mRNA from producing a protein showing that RNA molecules regulate the expression of genes

  3. Defining features Short length RNAs (~20-30 nucleotides) Association with Argonaute (Ago) family of proteins allowing reduced expression of target genes Through either mRNA degradation or translational repression Mechanism of RNA interference

  4. Guides sequence specific degradation of mRNAs homologous in sequence to the siRNAs Long dsRNA is cleaved by the Dicer enzyme complex into siRNA Approximately 21 bps in length and have a bp structure characterised by 2 nucleotide 3’overhangs Incorporated into AGO2 and the RNAi-induced silencing complex (RISC) RISC mediates unwinding of the siRNA duplex SS siRNA (antisense) that is coupled to RISC, binds to target mRNA Directs mRNA cleavage (catalytic domain of AGO2) siRNA pathway

  5. Pri-miRNA transcripts that are transcribed and processed by Drosha enzyme complex to create Pre-miRNA Exported to the cytoplasm and bind to Dicer enzyme complex Processed for AGO2-RISC complex RNA duplex loaded onto RISC has imperfect sequence complementarity, sense strand is unwound leaving mature miRNA Recognises target sites in 3’UTR (‘seed sequences’) and leads to direct translational inhibition Can also lead to mRNA target degradation in processing (P)-bodies. miRNA pathway

  6. Players in miRNA and siRNA • Argonaute (AGO) • PAZ domain binds the characteristic two-base 3' overhangs of siRNAs • PIWI domain: dsRNA guided hydrolysis of ssRNA • Slicer in RISC • Dicer (DCR) • Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA • TRBP • Cofactor for Dicer • RISC • RNA induced silencing complex

  7. Comparison of siRNA and miRNA properties

  8. Physiological roles of this process • siRNA • Silencing of ‘transposable elements’, repetitive genes and viruses. • Function for post transcriptional regulation or antiviral defence • miRNA • Encoded by genes in the host genome • Triggered by Pol II transcription (pri-mRNAs) • Function for regulation of mRNA stability and translation • Pre-miRNAs are sometimes produced by the nuclear pre-mRNA splicing pathway instead of Drosha processing • miRNAs regulate diverse cellular pathways and are widely believed to regulate most biological processes from housekeeping functions to responses to environmental stress

  9. Functional genomics • Valuable research tool • Inhibit gene expression in model organisms • Extremely useful for learning what genes do • Allow large-scale functional genomic analysis • Potent, long lasting post transcriptional silencing of specific genes • Large libraries of RNAi reagents can be used to knock down individual gene expression on a genome wide scale • Particularly useful in somatic mammalian cells • If exposed to dsRNA greater than 30bps cause interferon mediated innate immune response • Completion of human, mouse and rat genomes has provided a wealth of sequence information which can be used

  10. Example • Most functional genomic applications have used C.elegans and Drosophila • important models of development • dsRNA for the gene of interest is injected into the oocyte • Inactivates homologous gene and phenotype can be examined for clues to gene function

  11. RNAi based functional genomics • The selective ‘knock-down’ of genes has allowed dissection of molecular pathways • siRNA libraries have been used to study the role of proteins in:- • DNA damage response • Cell cycle control • DNA methylation • Cancer research

  12. RNAi can be triggered by two different pathways A) Synthetic effector siRNAs are delivered by various carriers to target cells B) plasmid based expression of short hairpin loops which give rise to siRNAs in vivo (based on nuclear synthesis of short hairpin RNAs (shRNAs) which are transported to the cytoplasm via the miRNA export pathway and are processed into siRNAs by Dicer). Methods of delivery

  13. Therapy for certain diseases • siRNAs have been shown to effectively down regulate gene expression in human cells • To control disease-associated genes makes RNAi an attractive choice for therapeutics • Cancer, autoimmune disease, dominant genetic disorders, viral infections • Research into efficient delivery agents • Liposomal carriers allow easy uptake by cell but don’t target specific cells • Aptamers, antibodies and peptides are useful for cell-type specific delivery • To date only a few candidate compounds have made it to clinical trials

  14. Human disease therapeutic applications • First used in 2004 in patients with age-related macular degeneration • Introduced siRNA by intravitreal injection to target the vascular endothelial growth factor (VEGF) mRNA • Eye allows siRNA to be delivered directly to the diseased tissue • Would not achieve endpoint of reducing vision loss • Huntington disease mouse model • Gain of function • RNAi was shown to improve motor and neuropathological abnormalities • Potential cancer treatment • Silencing genes differentially upregulated in tumour cells or genes involved in cell division

  15. Possible risks • Therapeutic siRNAs must reach their intended targets as might cause damage to nontargeted cells • Specificity and efficiency of siRNA • Toxicity • Study of liver disease in mice showed high fatalities possible due to ‘oversaturation’ of the dsRNA pathway • shRNAs are processed in the nucleus and exported to the cytoplasm utilising the cell mechanism • Thought to have been competing with miRNAs in the liver

  16. Further reading • Lares et al (2010) RNAi and small interfering RNAs in human disease therapeutic applications. Trends in Biotechnology 28 (11): 570-579. • Ghiildiyal and Zamore (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet. 10 (2): 94-108 • Kim and Rossi (2008) RNAi mechanisms and applications. Biotechniques 44 (5): 613-616.

  17. Essay Plan • RNAi controls gene expression • Post transcriptional silencing, cleavage/repression • Two pathways, siRNA - dsRNA, DICER, cleavage of specific mRNA • miRNA - from nucleus ssRNA, same pathway as siRNA, repression. Involved in many diff biological functions • Critical biological reagents, investigate gene function by silencing mRNA • Useful for gene therapy - gain of function mutations • Problems, never proven to work, interfere with pathways, might effect other mRNA function

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