1-Regulatory RNA, 2- RNA interference and micro RNA, 3-Retroviruses,

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TOPICS-I. 1-Regulatory RNA, 2- RNA interference and micro RNA, 3-Retroviruses, 4-Transposons and Retroposons, 5-Promoters and Enhancers , 6-Activating Transcription, 7-RNA Splicing and Processing, 8-Chromosomes-Nucleosomes, 9-Controlling Chromatin Remodeling and Structure.
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TOPICS-I1-Regulatory RNA, 2-RNA interference and micro RNA, 3-Retroviruses, 4-Transposons and Retroposons, 5-Promoters and Enhancers, 6-Activating Transcription, 7-RNA Splicing and Processing, 8-Chromosomes-Nucleosomes, 9-Controlling Chromatin Remodeling and Structure.TOPICS-II10-Gene Regulation I, 11-Gene Regulation II, 12-Protein Synthesis BooksMolecular Biology of the CellGenes IX/X/...Any....Presentation:Topic selection/studentRegulatory RNA (review and paper)RNA interferenceMicro RNATransposons, Retroposons and RetrovirusesPromoters and Enhancers Activating Transcription RNA Splicing and ProcessingControlling Chromatin Structure and Chromatin remodelingRetrovirusesGene RegulationEvaluation:1- Exam= 50% (Parts-I and II)2-Presentation 1=50%* (review and specific paper, idea, concept and experimental-selected by the instructor) 2a-presentation: 40% 2b-participation: 10%(*)-Each student will make a presentation for 15 min. (for specific papers) and 20 min (for review papers) with discussion (2-3 questions).Exam (Topics plus selected papers)Open/Close bookRegulatory RNAMicroRNAs Are Regulators in Many Eukaryotes
  • Animal and plant genomes code for many short (∼22 base) RNA molecules called microRNAs.
  • MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs.
  • C. elegans: regulator gene lin4 and its target gene lin14 (lin: Proteins for larval development)RNA Interference Is Related to Gene Silencing
  • RNA interference triggers degradation of mRNAs complementary to either strand of a short dsRNA.
  • Figure 13.21dsRNA may cause silencing of host genes.Figure 13.22What is RNA interference?Shooting down mRNARNAi
  • Background
  • What is it?
  • Why use it?
  • The mechanism and process
  • Experimental considerations
  • PlasmidVirusJorgensen 1990van der Krol 1990Gene injection (pigmentationEnzyme-petunias)Expectation: more red colorCo-suppression of transgene and endogenous gene.Hamilton and Baulcombe 1998Bill Douherty and Lindbo 1993Identification of short antisense RNA sequences dsRNA?How?Gene injection with a complete tobaccoetch virus particle.Expectation: virus expressionCo-suppression of transgene and virus particles via RNA.Ambros 1993 (2000)Fire and Mello 1998Identification of small RNA in C. elegans (micro RNA)Injection of dsRNA into C. elegansRNA interference (RNAi) or silencingShooting mRNA means RNA interferenceWhat is RNA interference?--Gene “knockdown”--A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why?--A way for the cell to defend itself.Why use RNAi?1. The most powerful way to inhibit gene expression and acquire info about the gene’s function fast2. Works in any cell/organism3. Uses conserved endogenous machinery4. Potent at low concentrations5. Highly specific.The mechanism of small interfering RNAs (siRNAs)What happens? dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA. The RNAi processRNA interference:--A type of gene regulation --Involve small RNA molecules --Induce a double stranded RNAStep 1
  • dsRNA is processed into sense and antisense RNAs
  • 21-25 nucleotides in length
  • have 2-3 nt 3’ overhanging ends
  • Done by Dicer (an RNase III-type enzyme)
  • The siRNAs associate with RISC (RNA- induced silencing complex) and unwind Step 2the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs. Step 3RISC cuts the mRNA approximately in the middle of the region paired with the siRNAThe mRNA is degraded furtherStep 4ssRNA (exogenous)RNA-dependentRNA polymeraseCatalysis: RdRP copies RNA making more ds RNA.Dicer complex: RNAase III with ATP hydrolysis requirement.Dicer cuts, unwinds dsRNAand generates more siRNA.More RdRP is activated and more dsRNA is made.RISC complex:RNA-InducibleSilencing Complex with ATPhydrolysis.(RdRP)(endogenous)Gene regulation by small RNAsDicer gene in C. eleganssiRNAs degrade mRNAto stop gene expression quicklySmall temporal (St) RNAs prevent translation to stop gene expression quickly-your RNAi?--MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression (down-regulation).--miRNAs are encoded by genes that are non-coding RNAs ( no proteins are made)--Stem-loop or hairpin loop intra-molecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. Experimental Considerations
  • Transfection method:
  • 1-Lipofectamine 2000--cationic lipids to bind siRNA and neutral
  • lipids to allow escape from Endosomes
  • 2-Plamids/Viruses--express small fragment of hairpin DNA
  • Transfection efficiency
  • Negative controls --scrambled siRNA
  • Off-target effects:
  • Sense (or antisense) strand is homologous to another sequence
  • Activation of stress response pathways “apoptosis”
  • http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.htmlhttp://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.htmlGrowth Factor Receptor Binding Protein (Grb) 2-mediatedRecruitment of the RING Domain of Cbl to the EpidermalGrowth Factor Receptor (EGFR) Is Essential and Sufficient toSupport Receptor EndocytosisFangtian Huang and Alexander SorkinKnockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediatedendocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFRendocytosis, we have generated cell lines in which endogenous Grb2 was replaced by a modified yellow fluorescent protein(YFP)-tagged Grb2 and it was expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect ofGrb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis.To generate HeLa cells stably expressing Grb2-YFP, endogenousGrb2 was knocked down using vector-based shorthairpin RNA (shRNA) with simultaneous expression ofGrb2-YFP that has silencing mutations rendering this constructinsensitive to shRNA.pSilencer1.0-U6 vector--pSuper-H1 vector--retrovirus//inducibleType III RNA pol III promoter ----U6 small nuclear promoter (U6) ----human H1 promoter (H1)
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