L5 RNA Mediated Regulation of Gene Expression.pdf

L5 RNA Mediated Regulation of Gene Expression Describe the origin of micro RNAs and be able to describe the mechanism that produces them in eukaryotic cell. Describe how miRNA regulates gene expression in eukaryote cells. Outline the domain structure of RNase III fami...

L5 RNA Mediated Regulation of Gene Expression Describe the origin of micro RNAs and be able to describe the mechanism that produces them in eukaryotic cell. Describe how miRNA regulates gene expression in eukaryote cells. Outline the domain structure of RNase III family members and explain how this relates to their function. Explain how miRNAs can be identified using miRNA arrays and miRNA sequencing. Outline the number of miRNA expressed by eukaryote genomes, the % of gene predicted to be regulated by miRNA in the human genome. Explain how alterations in miRNA sequence and expression are linked to human disease. Explain the reasons why it is thought RNAi mechanisms evolved. Explain how RNAi has been used to create novel mutant phenotypes in C. elegans for research reasons. Describe RNAi could be used in therapeutic way to treat human disease and infection. Describe the origin of micro RNAs and be able to describe the mechanism that produces them in eukaryotic cell. MicroRNAs (miRNAs) are small, non-coding RNA molecules that are transcribed from genomic DNA in eukaryotic cells. Mechanism of miRNA production The production of primary miRNAs (pri-miRNAs) is regulated by transcription factors, similar to other genes. Pri-miRNAs are often over 1000 nucleotides long and can produce more than one miRNA. The pri-miRNA undergoes processing in the nucleus by the microprocessor complex, which consists of the RNase III enzyme Drosha and the double- stranded RNA-binding protein DGCR8. Drosha cleaves the stem-loop structure of the pri-miRNA, releasing a shorter precursor miRNA (pre-miRNA) of around 60-70 nucleotides. The pre-miRNA is then exported to the cytoplasm by the Exportin-5 protein in an energy-dependent process (using GTP-Ran). The dicer enzyme cleaves the terminal loop of the pre-miRNA to create an miRNA:miRNA duplex 21-25 nucleotides in length Dicer induces formation of the RISC complex with an argonaute protein. The guide strand (antisense) is selected and loaded into Argonaute and the passenger (sense) strand is discarded. The mature RISC regulates gene expression. TRBP and DICER can dissociate from mature RISC after guide strand loading.  Typically the guide strand binds to the 3’ untranslated region (3’UTR) of its target mRNA target. In mammals the guide strand is typically only partially complementary to its target. Most common mammalian mechanisms 1. Deadenylation (removal 3’ polyA tail) followed by degradation. 2. Inhibition of translation Describe how miRNA regulates gene expression in eukaryote cells. RNA interference (RNAi) silences gene expression transcriptionally or post- transcriptionally. Small regulatory RNAs can bind to mRNA targets by complementary base pairing. This can lead to destruction of the target mRNA or can block its translation. Some regulatory RNAs can enter the nucleus and switch off gene transcription of the target gene by modifying chromatin structure. Mechanisms of target regulation by miRNAs Endonucleolytic cleavage Deadenylation and degradation o Removal of polyA tail. Inhibition of translation initiation o Argonaute and the guide strand stop the ribosome from binding to the translation initiation site (ATG). Inhibition after translation initiation o mRNA dissociates with subsequent loss of protein product. Stimulation of translation o Much rarer Outline the domain structure of RNase III family members and explain how this relates to their function. RNase III family members are nucleases that show specificity for double- stranded RNA. They typically contain one or two RNase III domains and one double-stranded RNA-binding domain (dsRBD). Nuclease: enzyme that degrades nucleic acids. DNase degrades DNA and RNase degrades RNA. Drosha and dicer involved in RNAi mediated gene expression They show specificity for double stranded RNA Three structural classes: Class I (prokaryote): Has 1 RNase III domain and 1 dsRNA-binding domain. Class II (eukaryote) Drosha: Has 2 RNase III domains and 1 dsRNA-binding domain. o Cleaves stem loop 11bp from junction Class III (eukaryote) Dicer: contains 2 RNase III domains and 1 dsRNA- binding domain (dsRBD) and an additional PAZ domain.  o PAZ domain binds to 5’ and 3’ ends of pre-miRNA hairpin and then the two RNase III domains cut the dsRNA stem (to remove the loop) and create the miRNA:miRNA* duplex which is 21-25 nucleotides in length. There is only 1 dicer and 1 drosha in humans, but up to 4 argonautes. Explain the reasons why it is thought RNAi mechanisms evolved. RNAi mechanisms are thought to have evolved for two main reasons: To protect the genome from the movement of transposable genetic elements, such as transposons and retroelements, which could disrupt gene function, and To provide a defence mechanism against viral infection by silencing viral RNAs. o This was discovered because introducing mutations to argounaute in plants makes them more susceptible to viruses. Explain how miRNAs can be identified using miRNA arrays and miRNA sequencing. miRNA sequencing 1. Purify miRNA from total RNA using size selection (25 nucleotides a. long). 2. Anneal a 3’ adaptor to 3’ end of miRNA  3. Anneal a 5’ adaptor to 3’ end of miRNA 4. Anneal reverse transcriptase primer to 3’ adapter 5. Add reverse transcriptase to synthesise rest of DNA strand 6. End result: single strand of DNA. Middle of strand is complementary sequence to miRNA. 7. Take single stranded cDNA and put it in a PCR reaction with 2 PCR primers 8. Use PCR to amplify single stranded DNA product of reverse transcription End result: Double stranded DNA product that has miRNA sequence in the middle. Known sequences at beginning and end (adapter and PCR primer). miRNA arrays to study expression profile. a. Synthetic oligonucleotides complementary to the mature miRNA are synthesized and spotted on slides (one dot = one miRNA). b. miRNA is purified from different tissues and the ends of the miRNA are labelled with a fluorescent dye. c. The labelled miRNA is hybridized to oligonucleotides on the slides and then the amount of fluorescence is measured. Outline the number of miRNA expressed by eukaryote genomes, the % of gene predicted to be regulated by miRNA in the human genome. The human genome is thought to encode over 2,000 microRNAs. 6 At least 60% of human genes are predicted to be regulated by microRNAs in some way. miRNAs have been shown to play important roles in the regulation of many biological processes and have been linked to a variety of human diseases, including cancer. Explain how alterations in miRNA sequence and expression are linked to human disease. Changes in miRNA expression levels and mutations within miRNA sequences have been associated with a number of human diseases. For example, a mutation in the miR-96 sequence causes hereditary progressive hearing loss, while a mutation in miR-184 leads to the eye disorder keratoconus and cataract. miRNA dysregulation due to mutation has been linked to many complex human diseases involving multiple gene products, such as various types of cancer. Associated with complex disesases where there are abnormalities in more than one gene product. miRNA often DOWNREGULATED in cancer. Explain how RNAi has been used to create novel mutant phenotypes in C. elegans for research reasons. Researchers have used RNAi in the model organism C. elegans to systematically "silence" the expression of individual genes and observe the resulting phenotypes. This has allowed the identification of many genes involved in processes like fat regulation, with potential relevance to human obesity. RNAi has been used to target nearly all C. elegans genes, creating a valuable resource for understanding gene function. Take gene of interest and clone it into a plasmid which can replicate in e. coli. It has 2 promotors on either side of the gene insert, which creates an mRNA which is the sense strand and antisense strand. Take the sense strand and transform it into E. coli. Spread E. coli on surface of agar plate, C. elegans eat e. coli as a food source. Score progeny for phenotype. Or: Harvest double stranded RNA Inject into gonads of nematode. Collect eggs, allow progeny to develop, score progeny. Take double stranded RNA, soak worms in dsRNA for 24 hours, transfer to agar plates, score progeny. Describe RNAi could be used in therapeutic way to treat human disease and infection. Research can be done to identify genes of interest e.g. obesity genes found in c. elegans. E.g. 50% of fat regulatory genes in c. elegans not previously implicated in fat storage. Using siRNA to treat human disease You can deliver RNAi based therapies by 1. Using chemically synthesised siRNA (small interfering RNA) which can be delivered by a number of techniques (see next slide) 2. Delivery of siRNA encoding genes using engineered viruses that will generate siRNA by transcription in target cells. siRNA = small interfering RNA – 20-25 base pairs – produced bycreating synthetic oligonucleotides and hybridizing them together. Ways of delivering siRNAs. Use nanoparticle – surround siRNA with lipid to allow it to enter the cell via endocytosis. Released into cytoplasm and binds to argonaute. Guide strand binds to target mRNA to lead to silencing effect. Attach to molecule – e.g. sugar conjugate – encourages cell to attach to cell surface receptors Use viral vector – siRNA is part of DNA in viral vector. Gene is expressed from plasmid. Single stranded RNA formed (short hairpin RNA). This is cleaved and exported from nucleus and goes into normal miRNA pathway. Possible uses 1. To ‘silence’ detrimental human genes e.g. those that cause cancer. 2. To ‘silence’ viruses e.g. HIV and HSV Example: Treatment for wet age related macular degeneration (Bevasiranib) downregulates VegF which causes overgrowth of blood vessels behind the retina.