2006??????? RNA interference

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2006???????RNA interference

• ???????
• ???????
• ?????B
• ?????(B9702086)
• ?????(B9702055)
• ???(B9702081)
• ???(B9702085)
• ???(B9702087)
• ???(B9702090)
• ???(B9702092)
• ???(B9702102)

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RNAi ????

• ?????microRNA??????
• ??????????
• ?????????

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Andrew Z. Fire and Craig C. Mello

• 1983-1986??Sydney Brenner?????????????
• ?????????????????
• 1982??Dr. David Hirsh??????????
• ????????????????????,????????????????

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??

• Fire, A., Albertson,D., Harrison, S.Moerman,D.
  Production of antisenseRNA leads to effective and
  specific inhibition of gene expression in C.
  elegans muscle. Development 113, 503514 (1991).
• Mello, C. Fire, A. DNA transformation. Methods
  Cell Biol. 48, 451482 (1995).
• Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas,
  S. E. Driver, and C. C. Mello. "Potent and
  Specific Genetic Interference by Double-stranded
  RNA in Caenorhabditis Elegans." Nature 391
  (1998) 806-811.

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RNAi ????

• 1990? (Rich Jorgensen?A. R. Stuitji)
• ???????????(co-suppression)
• 1992? (Macino??Cogoni )
• ?????(Neurospora crassa)?quelling??

?? chalcone synthase mRNA (CHS)
Wild-type
transgenic
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???????????

• 1993? (David Baulcombe)
• ??????(Tobacco etch virus)????????(PTGS)
  (??PVX)
• 1995? (Kenneth Kemphue Su Guo )
• ?sense?antisense RNA???? (??unc-22)

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????(??RNAi???)

• 1998? (Andrew Z. Fire Craig C. Mello )
• ?dsRNA????????? (??dsmex-3)

a???? b???? (?mex-3??) c??antisense-RNA
? d??dsRNA ? (??????)
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???

• ???????GFP?lacZ??DNA??????
• ??ds-gfpG RNA??ds-lacZL RNA

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????

• 1998?
• ?Nature????RNAi???
• 2000?
• RISC(RNA-inducing silencing complex)????????
• 2001?
• ??siRNA(short interfering RNA)??Dicer(??RNase)
• 2002?
• RNAi?????????,?HIV
• 2006?
• ???????????

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Post-transcriptional gene silencing

• NMD (Nonsense mediated decay)
• RNAi (RNA interference)

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RNAi (RNA interference)

• ?mRNA??(RNA degradation)?? mRNA????
• ???mRNA??????????

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RNAimechanism
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(No Transcript)
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siRNA

• ????siRNA???5?????siRNA???RNAi???,3?????2??????s
  iRNA????mRNA?????

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transposon

• ??????DNA??,?????????????????,?????????,??????????
  
• ???????????????????? RNA???????????????DNA????????
  ?????????????????RNA?????Dicer???????

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shRNA??
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miRNA??
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miRNA???

• miRNAs are encoded by genes from whose DNA they
  are transcribed but miRNA are not translated into
  protein
• animal miRNAs are usually complementary to a site
  in the 3' UTR
• plant miRNAs are usually complementary to coding
  regions of mRNAs

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miRNA shRNA
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miR-34 family function as tumor suppressors

• miR-34 family members are highly conserved during
  evolution
• miR-34a is located within chromosome 1p36 region,
  which is commonly deleted in human neuroblastoma
• Primary neuroblastomas and cell lines often
  showed low levels of miR-34a expression
• Forced expression of miR-34a in these cells
  inhibited proliferation and activated cell death
  pathways

Cancer Research (2007) 67 11099-11101
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(No Transcript)
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Dicer structure
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RISC (RNA-induced silencing complex)

• RISC ????????Argonaute???????
• ?????????PAZ?Mid?PIWI?N

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???????????
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siRNA ???????

• ???????,?????dsRNA???????mRNA??
• ????????
• ??dsRNA?Dicer??????? siRNA (primer)
• ?mRNA???,?primer,RdRP(RNA-dependent RNA
  polymerase )??????
• ?????siRNA???????
• ????

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(No Transcript)
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siRNA Design Guidelines

• General Design Guidelines
• 1. Find 21 nt sequences in the target mRNA that
  begin with an AA dinucleotide.
• 2. Select 2-4 target sequences.
• - GC content 30-50
• - 5-6 T's(terminate transcription)
• - avoid intron regions and stretches of 4 or
  more bases
• - perform BLAST homology search
• 3. Design appropriate controls.
• - negative control
• - Additional siRNA sequences targeting the same
  mRNA.

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(No Transcript)
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pSilencer adeno Recombination and Packaging in
HEK 293 Cells
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HeLa cells transfected with Cy3 labeled ß-Actin siRNA. Untransfected control. Red Cy3 labeled siRNA, Blue DAPI stained nuclei, Green(Immunofluorescence analysis ) ß-Actin protein.

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(No Transcript)
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treatment

• ??????
• CD4????
• ??????
• CCR5????
• ????
• CxCR4????
• ????????

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????????

• siRNA????????????????????
• Gag is a polyprotein and is an acronym for Group
  Antigens (ag).
• Pol is the reverse transcriptase.
• Env is the envelope protein.

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?????????

• HIV?1??
• ???siRNA?????????
• ????siRNA???????
• ???siRNA??HIV.1????????

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????

• Andrew Z. Fire Autobiography
• http//nobelprize.org/nobel_prizes/medicine/laurea
  tes/2006/fire-autobio.html
• Craig C. Mello Autobiography
• http//nobelprize.org/nobel_prizes/medicine/laurea
  tes/2006/mello-autobio.html
• ????2007?8?,416?,48 57?
• http//web1.nsc.gov.tw/public/Data/popsc/2007_111/
  48-57.pdf
• ????2008??????? 276-283
• http//www.idealversion.com/biomedicine/archives/0
  8v1n3/bm-08-18rr.pdf
• ??????????????
• http//homepage.ntu.edu.tw/epidem/miRNAs.pdf
• ????
• http//www.ncbi.nlm.nih.gov/pubmed/12042869?ordina
  lpos3itoolEntrezSystem2.PEntrez.Pubmed.Pubmed_R
  esultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDoc
  Sum
• http//nobelprize.org/nobel_prizes/medicine/laurea
  tes/2006/adv.html
• ??
• http//nobelprize.org/nobel_prizes/medicine/laurea
  tes/2006/info_en.pdf
• http//nobelprize.org/nobel_prizes/medicine/laurea
  tes/2006/adv.html
• http//en.wikipedia.org/wiki/Nonsense_mediated_dec
  ay
• RNAi??
• http//www.ncbi.nlm.nih.gov/genome/probe/doc/TechR
  nai.shtml

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????

• miRNA
• http//www.dyjzbjb.com/083ZS9.htm
• http//en.wikipedia.org/wiki/MicroRNA
• http//memo.cgu.edu.tw/shu-er/94E4B88AE5ADB8
  E69C9FE699AEE7949FE5A0B1E5918AE4B
  8BBE9A18C/Final20reports/A6.pdf
• http//rnajournal.cshlp.org/content/13/9/1402.full
  
• http//www.unimed.com.tw/pdf/08Q220SBI.pdf
• shRNA
• http//en.wikipedia.org/wiki/Small_hairpin_RNA
• DicerRISC
• Structural Basis for Double-Stranded RNA
  Processing by Dicer
• Ian J. MacRae,1,3 Kaihong Zhou,1,3 Fei Li,1
  Adrian Repic,1 Angela N. Brooks,1 W. Zacheus
  Cande,1 Paul D. Adams,4 Jennifer A.
  Doudna1,2,3,4
• Zamore P, Tuschl T, Sharp P, Bartel D (2000).
  "RNAi double-stranded RNA directs the
  ATP-dependent cleavage of mRNA at 21 to 23
  nucleotide intervals". Cell 101 (1) 2533.
• Vermeulen A, Behlen L, Reynolds A, Wolfson A,
  Marshall W, Karpilow J, Khvorova A (2005). "The
  contributions of dsRNA structure to dicer
  specificity and efficiency". RNA 11
• ??????
• http//www.biox.cn/content/20050531/14191.htm
• http//www.nature.com/focus/rnai/animations/animat
  ion/animation.htm
• ??
• http//www.ambion.com/techlib/tb/tb_506.html
• http//www.genscript.com/siRNA_technology.html

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????