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1-Regulatory RNA / CRISPR- Cas system/ RNA interference and micro RNA, 2-Retroviruses,

TOPICS. 1-Regulatory RNA / CRISPR- Cas system/ RNA interference and micro RNA, 2-Retroviruses, 3-Transposons and Retroposons , 4-Promoters and Enhancers , 5-Activating Transcription/ Epigenetic Effects 6-RNA Splicing and Processing / Enhancer: eRNAs and lncRNAs

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1-Regulatory RNA / CRISPR- Cas system/ RNA interference and micro RNA, 2-Retroviruses,

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  1. TOPICS 1-Regulatory RNA/CRISPR-Cas system/RNA interference and micro RNA, 2-Retroviruses, 3-Transposons and Retroposons, 4-Promoters and Enhancers, 5-Activating Transcription/Epigenetic Effects 6-RNA Splicing and Processing/Enhancer: eRNAs and lncRNAs 7-Chromosomes-Nucleosomes, 8-Controlling Chromatin Remodeling and Structure. 9-Gene Regulation I, 10-Gene Regulation II

  2. Books

  3. Books https://www.ncbi.nlm.nih.gov/pubmed/…. books

  4. Presentation:Topic selection/student Regulatory RNA (paper), RNA interference Micro RNA, Transposons, Retroposons and Retroviruses Promoters and Enhancers , Activating Transcription RNA Splicing and Processing, Controlling Chromatin Structure and Chromatin remodeling Retroviruses, Prokaryotic Gene Regulation-CRISPR-Cas 9 system The Mechanisms and Significance of mRNA Degradation RNA Splicing- SR Proteins

  5. Evaluation: 1- Exam= 50% 2-Reading Material= 20 % 3-Presentation =20% 4-Participation = 10 %* (*)-Each student will make a presentation for 20-25 min with 5 to 10 min discussion (2-3 questions).

  6. Exam Close book exam or Open book “Take home” exam

  7. Exam: 50%. -Exam will be “open” and it will have 25 “essay type” questions and each question will have 3 to 5 sub-questions. --Each correct answered question is equally weighted (2 points per correct question). --Exam date and time: It will start at 12:01:00 am on 10/30/17 and it will finish at 11:59:00 pm on 11/5/17. --No make up Exam will be given, except in cases of unforeseeable and severe documented circumstances beyond your control.

  8. Reading Material: 20%. --Outside of some lectures by the instructors, the course will focus on review articles for discussion (reading material). --Each student will write a specific “highlights” of the review paper assigned for each lecture. --The language of each “highlights” should be clear, concise and to the point, and you should use 5 full sentences and each sentence should contain no more then 90 characters. --Each “highlights” is equally weighted (2.22 points per correct presented “highlights”). --Highlights deadline: Each highlight has a specific deadline and they can NOT be sent via email. Only hard copy will be accepted. --No make up Highlight will be given, except in cases of unforeseeable and severe documented circumstances beyond your control.

  9. Highlights Deadline (12:30 pm) Genome Engineering 9/5/17 Retrotransposons and genome regulation 9/12/17 Transcription units 9/19/17 Chromatin and Enhancer 9/26/17 Nuclear organization 10/3/17 Nucleosome Structure and Function 10/10/17 mRNA degradation and splicing 10/17/17 The type II secretion system 10/24/17 The type III secretion system 10/31/17

  10. Presentations: 20% and Participation: 10%. --Students will choose original research or specific review articles that have been published in 2017 on selected topic assigned by the instructor (see specific topics section in the syllabus section). --Students must send 5 articles to the instructor, who will select the article to be presented no later than 9/19/17. The Instructor will then send the selected article for each student no later then 9/26/17 together with the exact day of the presentation.

  11. --Before each presentation (24 hrs), the presenter has to email to the whole class and instructor a ppt/pdf file of the PowerPoint presentation along with research paper. Each student will make a presentation (~25 minutes) on the research articles. --Preparation for the presentation involves knowing well the article as well as any supplemental material. ---Presentation of the data should involve getting to know how the experiments were done so that data can be critically analyzed.

  12. Regulatory RNA

  13. MicroRNAs 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)

  14. RNA Interference Is Related to Gene Silencing • RNA interference triggers degradation of mRNAs complementary to either strand of a short dsRNA. Figure 13.21

  15. dsRNA may cause silencing of host genes: no more proteins NK, p38, NFkB, PP2A Stress Signal 2’, 5’-oligoadenylate synthetase (2’, 5’-AS)

  16. What is RNA interference? Shooting down mRNA

  17. RNAi • Background • What is it? • Why use it? • The mechanism and process • Experimental considerations

  18. Plasmid Virus

  19. Jorgensen 1990 van der Krol 1990 Gene injection (pigmentation Enzyme-petunias) Expectation: more red color Co-suppression of transgene and endogenous gene. Hamilton and Baulcombe 1998 Bill Douherty and Lindbo 1993 Identification of short antisense RNA sequences dsRNA? How? Gene injection with a complete tobacco etch virus particle. Expectation: virus expression Co-suppression of transgene and virus particles via RNA. Ambros 1993 (2000) Fire and Mello 1998 Identification of small RNA in C. elegans (micro RNA) Injection of dsRNA into C. elegans RNA interference (RNAi) or silencing

  20. Shooting mRNA means RNA interference

  21. What 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.

  22. Why use RNAi? 1. The most powerful way to inhibit gene expression and acquire info about the gene’s function fast 2. Works in any cell/organism 3. Uses conserved endogenous machinery 4. Potent at low concentrations 5. Highly specific.

  23. The mechanism of small interfering RNAs (siRNAs) What happens? dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA.

  24. The RNAi process RNA interference: --A type of gene regulation --Involve small RNA molecules --Induce a double stranded RNA

  25. Step 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)

  26. The siRNAs associate with RISC (RNA- induced silencing complex) and unwind Step 2

  27. the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs. Step 3

  28. RISC cuts the mRNA approximately in the middle of the region paired with the siRNA The mRNA is degraded further Step 4

  29. ssRNA (exogenous) Exam RNA-dependent RNA polymerase Catalysis: RdRP copies RNA making more ds RNA. Dicer complex: RNAase III with ATP hydrolysis requirement. Dicer cuts, unwinds dsRNA and generates more siRNA. More RdRP is activated and more dsRNA is made. RISC complex:RNA-Inducible Silencing Complex with ATP hydrolysis. (RdRP) Exam (endogenous) SiRNP: small interfering ribonucleoprotein particle

  30. Gene regulation by small RNAs Dicer gene in C. elegans siRNAs degrade mRNA to stop gene expression quickly Small temporal (St) RNAs prevent translation to stop gene expression quickly

  31. -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.

  32. 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”

  33. 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.html

  34. Growth Factor Receptor Binding Protein (Grb) 2-mediated Recruitment of the RING Domain of Cbl to the Epidermal Growth Factor Receptor (EGFR) Is Essential and Sufficient to Support Receptor Endocytosis Fangtian Huang and Alexander Sorkin http://www.ncbi.nlm.nih.gov/pubmed/15635092

  35. Knockdown of Grb-2 by RNAi inhibited internalization of EGFR . We have generated cell lines in which endogenous Grb2 was replaced by a modified (YFP)-tagged Grb2 YFP-Grb2 was expressed at the physiological level Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis

  36. To generate HeLa cells stably expressing Grb2-YFP, endogenous Grb2 was knocked down using vector-based short hairpin RNA (shRNA) with simultaneous expression of Grb2-YFP that has silencing mutations rendering this construct insensitive to shRNA.

  37. pSilencer1.0-U6 vector-- pSuper-H1 vector-- retrovirus//inducible Type III RNA pol III promoter ----U6 small nuclear promoter (U6) ----human H1 promoter (H1)

  38. Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc. Xu D, Dwyer J, Li H, Duan W, Liu JP. http://www.ncbi.nlm.nih.gov/pubmed/18586674

  39. Philadelphia chromosome: chronic myelogenous leukemia (CML). http://www.ncbi.nlm.nih.gov/pubmed/20537386 ABL: kinase, BCR: Substrate

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