1 / 17

Tongju Li

Tongju Li. RNA Interference and Application in Channel Research. RNA interference. RNA interference is a conserved gene silencing mechanism that recognizes double-stranded RNA as a signal to trigger the sequence-specific degradation of homologous mRNA.

chaela
Download Presentation

Tongju Li

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Tongju Li RNA Interference and Application in Channel Research

  2. RNA interference • RNA interference is a conserved gene silencing mechanism that recognizes double-stranded RNA as a signal to trigger the sequence-specific degradation of homologous mRNA. • Orgnisms exhibiting RNAi-related phnomena including: • Kindom Species stage tested • Protozoans T Brucei Procyclic forms • P falciparum blood stage • Paramecium Mature form • Invertebrates C elegans Larval stage and adult stage • Vertebrates Zebra fish Embryo • X lasevis Embryo • Mice Prenatal, embryonic stages, and adult • Humans Human cell line • Plants Monocots/dicots siRNA/transgenics • Fungi N crassa Filamentous fungi • Algae C reinhardtii • Have been described as cosuppression or PTGS (post transcriptional silencing) in plants, quelling in fungi, and RNAi in the animal kingdom. • Functions: Protection of the genome against invasion by mobile genetic elements such as viruses and transposons as well as orchestrated functioning of the developmental programs of eukaryotic organisms. A B RNA-interference in C elegans. The transgenic line carries gfp reporter constructs. A: animals injected with a control dsRNA; B: animals injected with dsRNA corresponding to GFP gene. FIREA et al. Nature391, 806 - 811 (1998); . Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

  3. Mechanisms: Degradation Dicer a double-strand-specific RNAse. Diser contains two RNAse III domains, a dsRNA binding motif , ATPase domain and a PAZ motif. siRNA Small interference RNA. 21-23 nt with symmetric 2-3-nt 3‘ overhangs and 5‘ phosphate and 3‘ hydroxyl groups Effector in the RNAi. RISC RNA-inducing silencing complex. Contanins PAZ domain through which it may interact with Dicer. Click here to download/view a movie describing the process from http://www.sirna.com

  4. Amplification & RISC-independent mRNA degradation Left: Amplification of siRNA and the RISC-independent mRNA degradation in Drosophila. the formed siRNA formed through dsRNA digestion are postulated to function as primers for the target RNA-templated synthesis of cRNA by an RdRP. The resulting dsRNA is then proposed to be cleaved by Dicer into a new crop of siRNAs, which can prime the conversion of additional target RNAs into dsRNA. Right: RISC-dependent mRNA degradation. RdRp:RNA-dependent RNA polymerase. Play an essential role in RNAi in plants, N crassa, and C elegans. However, no RdRps have been found in humans and flies. Dianne S et al. Molecular Cell, Vol. 10, 537–548 Evidence that siRNAs Function as Guides, Not Primers, in the Drosophila and Human RNAi Pathways

  5. Mechanism: Transition Transitive RNAi in C elegans. The transgenic line carries two different gfp reporter constructs (pSAK2 and pSAK4). Animals injected with gfp dsRNA show near-complete (98%) loss of both nuclear and mitochondrial GFP. T Sijen et al. Cell, Vol. 107, 465–476,2001 On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing

  6. dsRNA as inducer • Viral dsRNA (adenovirus, hsv, influenza etc) • Exogenous ds RNA • Transposon dsRNA • Endogenous dsRNA • Trig RNAi (left). • Activate protein kinase R which inactivate translation factor eIF2a, leading to a global suppression of protein synthesis and subsequently to programmed cell death. • Activate 2'–5' oligoadenylate synthetases leading to ATP polymerization, endonuclease RNase L activation, resulting in the cleavage of several RNA species including ribosomal RNA, then an inhibition of mRNA translation. RNA Pol II based, allow inducible , tissue or cell type specific RNA expression Arenz C and Schepers U. Naturwissenschaften (203) 90:345-359

  7. siRNA design • Sequence motifs: AAN19TT, NAN19NN, NARN17YNN and NANN17YNN (where N is any nucleotide, R is a purine and Y is a pyrimidine). • 50% GC content (30–70%). • Any region of mRNA sites for mRNA-binding proteins in the 5' UTR, 3' UTR, start codon or exon–exon boundaries, repetitive sequences should be avoided. • Avoid stretches of > 4 T‘s or A‘s in the target sequence when designing sequences to be expressed from an RNA Plo III promoter. • Several siRNAs to a gene and validate the efficiency of each. • Negtive control siRNA, which can have the same nucleotide composition as the your siRNA but lack significant sequence homology to the genome. • No significant homology with other genes.

  8. siRNA generation Dicer TTTT UUUU in vitro • Chemical synthesis • dsRNA digestion in vivo • PCR product • Plasmid • viral vectors (Oncoretrovirus; Lentivirus; adenovirus)

  9. Plasmide

  10. Adenovirus vector

  11. HIV Strategies for targeting viruses in mammalian cells Silva JM , Trends Mol Med. 2002 Nov;8(11):505-8. RNA interference: a promising approach to antiviral therapy?

  12. K+ Channel Dissection Dissection of voltage-dependent components by RNAi. • Family of K+ currents obtained from a slo-2 mutant muscle cell showing only the voltage-dependent components. • Family of K+ current traces for SHAL currents (slo-2 mutant cell treated with Shaker RNAi). • SHAKER currents (a slo-2 mutant cell treated with Shal RNAi). Cells were held at -70 mV and stepped from -70 to +60 mV in 10-mV increments. Santi CM et al. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14391-6.Dissection of K+ currents in Caenorhabditis elegans muscle cells by genetics and RNA interference.

  13. Aquaporin AQP4 and GFAP expression during AQP4 gene silencing. Astrocytes were analyzed in parallel for AQP4 (red) and GFAP (green) expression 2, 4, and 6 days after treatment with CTRL siRNA and AQP4 siRNA. Nicchia GP et al. FASEB J. 2003 Aug;17(11):1508-10. Epub 2003 Jun 17. Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes.

  14. TRPM7 Suppression of IOGD and TRPM-like currents in cultured neurons by RNAi. • recordings of enhanced IOGD currents in controls and chemically anoxic neurons. • IOGD currents and single-cell PCR from chemically anoxic cultured neurons treated with control siRNA or siRNAT1. • effect of inhibiting TRPM7 by RNAi on the enhancement PIP2-induced currents. Aarts M et al. Cell. 2003 Dec 26;115(7):863-77. A key role for TRPM7 channels in anoxic neuronal death.

  15. TRPC1 linked to the regulation of cell volume Evidence that TRPC1 forms a Ca2+-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1 The TRPC1 (transient receptor potential canonical 1) protein is thought to encode a non-selective cation channel..... The decrease in endo-genous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. ...... The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained J CHEN and G J. BARRITT. Biochem. J. (2003) 373 (327–336) Treatment with siRNA targeted against TRPC1 enhances the increase in cell volume and the regulatory cell volume decrease when the incubation medium is changed from a normal to a hypotonic medium H4-IIE cells (derived from a rat liver hepatoma) were transfected with chemical sythesised TRPC1 siRNA or were mock-transfected, incubated for 48–72 h, then transferred to normal or hypotonic medium and cell size measured.

  16. IP3 Receptor and Ca2+ signaling Distinct Roles of Inositol 1,4,5-Trisphosphate Receptor Types 1 and 3 in Ca2+ Signaling ....HeLa cells express comparableamounts of IP3R1 and IP3R3, but knockdown by RNAi of each subtype results in dramatically distinct Ca2+ signalingpatterns. ..... Effects of IP3R3 knockdown are surprisinglysimilar in COS-7 cells......Therefore, differentialexpression of the IP3R subtype is critical for various formsof Ca2+ signaling, and, particularly, IP3R1 and IP3R3 have oppositeroles in generating Ca2+ oscillations. Effects of IP3R knock-down on ATP-induced [Ca2+]i signaling in HeLa cells in the absence of extracellular Ca2+. M Hattori et al. JBC, Vol. 279:11967-11975, 2004

  17. Nedd4 and ENaC Relative Contribution of Nedd4 and Nedd4-2 to ENaC Regulation in Epithelia Determined by RNAi Epithelial Na+ transport is regulated in large part by mechanismsthat control expression of the epithelial Na+ channel (ENaC)at the cell surface. Nedd4 and Nedd4-2 are candidates to controlENaC surface expression..... ......in two epithelial celllines (Fischer rat thyroid and H441), small interfering RNA(siRNA) against Nedd4-2 increased amiloride-sensitive Na+ current, but Nedd4 siRNA did not. A mutationassociated with Liddle's syndrome ( R566X) abolished the effectof Nedd4-2 siRNA, suggesting that a defect in ENaC regulationby Nedd4-2 contributes to the pathogenesis of this inheritedform of hypertension. ....Thus, endogenous Nedd4-2 negatively regulates ENaC in epitheliaand is a component of a signaling pathway.... PM Snyder et al. JBC, Vol. 279:5042-5046, 2004 Silencing of endogenous Nedd4 and Nedd4-2.

More Related