中央研究院生物医学研究所

Micro-RNA Processing Pathway
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miRNAs are endogenous ~22 nt singlestranded noncoding RNAs Over 1000 miRNAs exist in humans. Primary miRNA transcribed by RNA polymerase II, with 5’-capped and 3’polyA and several thousand nt in length. The imperfect stem-loop structure in pri-miRNA recognized by the nuclear microprocessor complex (Drosha, DGCR8), which cleaves the stem ~22 bp away from the loop to release premiRNA. Pre-miRNA is an ~60 nt RNA hairpin intermediate with ~2nt 3’-overhang
{Ambros (2004) Nature 431:350}
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miRNAs and Their Functions
{Bartel (2004) Cell 116:281}
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ØMinimize
activating interferon response ØMinimize homology to irrelevant host mRNAs
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Hale Waihona Puke shRNAs should not contain extraneous inverted repeats.
{Cullen (2006) Gene Ther 13:503}
shRNA Utilize shRNA Utilize miRNA Processing miRNA Processing Machinery Machinery
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Exportin 5/GTP-Ran recognize small RNA stem-loops with ~2nt 3’-overhang, and transports them to cytoplasm Dicer binds to ~2nt 3’-overhang and cleaves 21-22 nt from the hairpin on both strands.
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Dicer (Dcr-2) and R2D2 recruit Ago2 Ago2 exchanges with Dcr-2/R2D2 by protein-protein interaction
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Ago2 cleaves the passenger strand. The mature RISC is ready for its target mRNA
shRNA Design And Target Selection
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Ensure efficient loading of RISC by the antisense siRNA strand. Design potent siRNA in inducing target mRNA degradation. Select an mRNA target available for RISC binding. Avoid “off-target” effects
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lin-4 miRNA targets the 3’ UTR of lin-14 and lin28 genes. In C. elegans, lin-4 miRNA coordinates downregulation of LIN-14 and LIN-28 proteins, involving stage-specific development.
1995}
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dsRNA mediated gene silencing in other organisms:
Ø Posttranscriptional
Trends Biotechnol 1990}
gene silencing in plants
{Jorgensen,
Ø Quelling
in fungus {Romano, Mol Microbiol 1992}
Mature miRNA in red
{Bartel (2004) Cell 116:281}
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Pre-miRNA
Imprecise Base Pairing of miRNAs with Their Targets
{Cullen (2006) Gene Ther 13:503}
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Structures Required for Nuclear Exportation And Dicer Processing
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Nuclear export of shRNAs and pre-miRNAs mediated by Exp5/Ran:GTP, which requires:
ØAn
RNA stem of 16 bp ØA terminal loop of >6 bp ØA short 3’ overhang
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shRNA Transcription And Processing
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5’ 3’-UU
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Transcription of shRNA usually by RNA polymerase III promoters, H1 and U6 promoters. U6 promoter strongly favors a “G” residue at the first position of transcript, while H1 promoter has a weak preference for an “A” residue. Transcription terminates after the second residue of a “TTTTT” stretch, resulting the 3’-terminal “UU” overhang.
{Cullen (2006) Gene Ther 13:503}
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shRNA Utilize shRNA Utilize miRNA Processing miRNA Processing Machinery Machinery
2006年7月21日
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Discovery of RNA Interference
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First report in animal cells: Injection of dsRNA in worm (C elegans) silenced genes with complementary sequence. {Fire, Development 1991; Guo, Cell
RNA Interference And Its Application for Chronic Hepatitis B Therapy
陶秘華 (Mi-Hua Tao, Ph.D.) 中央研究院生物醫學研究所 Institute of Biomedical Sciences Academia Sinica
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RNA Silencing RNA Silencing Pathways Pathways
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{Hannon & Rossi (2004) Nature 431:371}
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Tuschl and colleagues reported RNAi also occurred in mammalian cells. {Elbashir, Nature 2001} RNAi is a universal conserved mechanism in eukaryotic cells.
Long dsRNA and miRNA precursors are processed by Dicer to generate small (~22bp) dsRNA with symmetric 2-3-nt 3’ overhang, and 5’phosphate and 3’-OH The guide (antisense) strand incorporated into RISC; the passenger (sense) strand is released and degraded Activated RISC can direct mRNA cleavage, translational repression or induce chromatin modification
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Exportin 5/GTP-Ran recognizes small RNA stem-loops with ~2nt 3’-overhang, transports them to cytoplasm Dicer binds to ~2nt 3’overhang and cleaves 21-22 nt from the hairpin on both strands. Artificial shRNAs are transcribed by RNA polymerase III, with ~2nt 3’-overhang