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14 Cards in this Set
- Front
- Back
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1) Describe microRNAs and endogenous RNAs s7 |
1) -> microRNA: important in gene regulation, interact with Argonaute protein. -> endogenous RNAs: processed to smaller molecules, they interact with processing proteins like dicer and transport rna proteins like argonauts -> Can get from endogenous and exogenous sources |
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1) How can you induce heritable effects in the C elegans model? 2) What two argonaute proteins did the experiment study in the RNAi pathway? s8 |
1) Can change chromatin modifications either by introducing nuclear RNAi or by manipulation of chromatin modifiers, producing heritable effects. 2) -> HRDE-1 (heritable RNAi deficient-1) -> CSR-1 (chromosome segregation and RNAi deficient-1) both shown before to be associated with heritable small RNAs in the C. elegans germline of C. elegans and are required for RNAi inhertiance. |
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1) Why is C elegans a good model to use in this experiment? What work has been done before? 2) What are some challenges observed in the C elegans model and how can this be countered? s9 |
1) Can manipulate exogenous forms of dsRNA easily; can either directly injecting into the worm, or by feeding it, or take a bath of dsRNA and place the worms in it. These will then interact with dicer and argonaute proteins, triggering heritable RNAi responses 2) Effects generally get lost after 1-4 generations by itself. However, if you select for worms that exhibit silencing and cross these worms; can persist for more than 80 generations |
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1) What mystery led to the hypothesis of experiment? s10 |
1) The RNA agent required for RNAi inheritance, was diluted from in every generation, until reaching levels too low for gene expression. However, passive dilution of a limited RNA agent can't explain dynamics of RNAi inheritance decay. Dilution factor of about 4 billion after 4 gens seems to be too high |
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1) What was the rationale for the study and what was the goal? s11 |
1) Unclear why epigenetic responses become less effected at a certain rate. Unknown why RNAi inheritance decays. -> Recently found feedforward reaction that was shown to amplify secondary small RNAs to guide additional round of amplification, which lead to stable silencing of certain silencing responses that are established in the germline by piRNAs in C. elegans. |
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1) What did they discover in the model with RNA-seq? s12 |
1) Abundant endo-siRNAs that are heritable and even enriched in future generations. They align into the antisense orientation to multiple endo-siRNA biogenesis genes. Hypothesized that this may be the mechanism heritable small RNAs and regulated RNAi inheritance and biosynthesis genes for controlling expression |
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1) Describe the model system that they used s13 |
1) Agar plate with c elegans on top of the bacterial colony. They will eat bacteria as they go along. You can put different type of genes e.g. dsRNA antisense to GFP in the bacteria. The worms contained contained Ppie-1 gfp gene in the nuclei which drives GFP expression in the germ cells. -> First group had bacteria with anti-gfp -> second group: control with empty vector bacteria |
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1) What were the inital results of the bacteria experiment s14 |
1) Once the c elegans eat the dsRNA anti-gfp, and GFP expression stops. GFP expression continues with the control group. -> the inherited RNAi effect dissapeared after 4 gens |
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1) What did they do in the second stage of the experiment and what were the results? 2) What can be an explanation for the results? s15,16 |
1) Take the progeny of the first gen and was transferred to plates with bacteria with either empty vector or other dsRNA triggers (not anti-GFP) -> progeny exposed to secondary dsRNA triggers had more decrease in GFP expression than the empty vectors, even though no additional anti-GFP was added. 2) an RNAi response that targets a particular gene can extend the duration of an ancestoral heritable RNAi response, aimed against a different and unrelated gene |
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1) What do these secondary dsRNA triggers target and what was found next? s18 |
1) triggered somatically expressed genes e.g. dpy2, and targeted germline-expressed genes e.g Pdpy-30::mcherry -> authors then compared 11 different dsRNA second triggers which target genes that function in different cellular processes, and dont affect GFP levels on their own -> all the second triggers extended trans-generational duration of ancestral heritable silencing responses aimed against gfp. |
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1) Describe the potency analysis s19 |
1) anti-dpy-2 second dsRNA triggers found to be more potent than anti-mCherry in enhancing RNAi responses. They compared different triggers and found that while all the second dsRNA triggers were effective, targeting certain genes produced stronger effect. |
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1) What did they found when they measured for the original response? s20 |
1) Even with secondary trigger, found species of RNAi that you had against GFP that was found in the primary response in the first generation. |
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1) So what was the final proposed model that they put out? s21 |
1) -> initiation of an RNAi response turns on the exo-SiRNA inheritance system -> enhancing the production of exo-siRNAs at the expense of endogenous small RNA populations (and by altering the balance CSR-1 and HRDE-1 endo-siRNAs) -> A feedback response returns the siRNA inheritance mechanism back to OFF state by altering the regulation of endo-siRNAs on genes required for the inheritance of endo-siRNAs -> A transgenerational timer is being reset by initiation of new RNAi responses, and second triggers extend the inheritance of ancestral silencing |
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1) What are some future directions s22 |
1) interaction of RNAs and other epigenetic mechanisms e.g. chromatin mods, DNA mark -> higher organisms -> adaptive control over duration of environmental responses could affect evolution |
