RNAiA review of RNAi.
Edited by: Suzanne Kennedy and Nick Oswald"an essential book ... a valuable tool to all those interested in PCR" (Doodys); "an essential guide" Aus. J. Med. Sci. read more ...
Control, optimize and troubleshoot PCR, reverse transcriptase PCR, real-time PCR and quantitative PCR. An essential book.
RNAiAdapted from Premlata Shankar and Judy Lieberman in HIV Chemotherapy
RNAi: RNAi is a form of post-transcriptional gene regulation in which double-stranded RNA (dsRNA) molecules called small interfering RNA (siRNA) mediate sequence specific degradation of homologous mRNA. The initial step in RNAi is the cleavage of long double-stranded RNA into small, 21-23 mer siRNAs by an RNase III-like enzyme called Dicer. Each strand of the duplex siRNA product contains a 5' phosphate and a free 3' hydroxyl group; typically the two strands pair up for 19 base pairs and have 2- or 3-nt 3' unpaired overhangs. Exogenously introduced synthetic duplex RNAs with biochemical structure resembling Dicer-processed dsRNA can also effectively mediate RNAi. The Dicer enzyme also produces another class of regulatory RNA molecules called microRNAs (miRNAs) that are processed from endogenously generated hairpin structures. miRNAs act by inhibiting translation of RNA into protein, often by binding to the 3' untranslated regions of mRNAs. miRNAs also are involved in maintaining silenced regions of chromatin. Apart from their distinct modes of action, miRNA differ from siRNA in the less stringent requirement for sequence complementarity between the two strands. In some species, more than one Dicer enzyme may be responsible for generating different types of miRNAs. How do siRNAs lead to RNA degradation? Although the molecular details are still being worked out, a model has emerged. Duplex siRNAs are unwound by an ATP-dependent helicase and one strand is incorporated into a large multiprotein nuclease complex known as the RNA-induced silencing complex (RISC). Incorporation of small RNA activates RISC. Guided by the antisense strand of siRNA, the Argonaute2 endonuclease in the activated RISC recognizes and cleaves the corresponding mRNA, with a resultant decrease in the steady-state levels of mRNA. Argonaute proteins contain a conserved protein-protein interaction domain called PAZ and another conserved domain (PIWI) of uncertain function . Four proteins have so far been identified as candidate components of the RISC complex in mammalian cells. These are the Argonaute proteins eIF2C1 and eIF2C2 and gemin 3 (also known as DDX20, DEAD/H box polypeptide 20) and gemin 4, but their roles are not understood. The RNAi response is an intrinsic defense mechanism for combating viruses and protecting the genome from invasion by endogenously generated transposons. The notion of homology-directed gene silencing as an anti-viral response derives from a large body of data obtained in plants. Most plant viruses have double-stranded RNA genomes. RNAi is induced in response to replicative stage double-stranded RNA intermediates in the viral life cycle. Arabidopsis mutants that are defective in genes involved in RNAi are highly susceptible to cucumovirus and tobacco mosaic viral infection. The importance of RNAi in antiviral defense is also suggested by the fact that plant and insect viruses have developed counter defensive strategies, encoding proteins that suppress different steps in RNA silencing. However, an innate RNAi antiviral response has not yet been reported in mammals. This may be because evolution has endowed mammals with alternative antiviral mechanisms that override the RNAi response. Introduction of dsRNA larger than 30 nt into mammalian cells results in global repression of gene expression because of the activation of protein kinase PKR and 2', 5'-oligoadenylate synthetase that form part of the interferon (IFN) defense pathway against viruses. Nevertheless, the RNAi pathway is functionally intact in mammalian cells. Exogenously introduced siRNAs guide degradation of cognate mRNA and abrogate gene expression in an exquisitely sequence-specific manner. The important discovery that RNAi works in mammalian cells was soon followed by a flurry of research papers demonstrating the antiviral potential of this powerful new technology against a multitude of mammalian viruses including HIV-1 . RNAi is also being used in basic HIV research to silence host genes to understand better the interaction of HIV with host genes during the viral life cycle.
- Human Pathogenic Fungi
- Applied RNAi
- Molecular Diagnostics
- Phage Therapy
- Bioinformatics and Data Analysis in Microbiology
- The Cell Biology of Cyanobacteria
- Pathogenic Escherichia coli
- Campylobacter Ecology and Evolution
- Next-generation Sequencing
- Omics in Soil Science
- Applications of Molecular Microbiological Methods
- Genome Analysis
- Bacterial Toxins
- Bacterial Membranes
- Cold-Adapted Microorganisms
- RNA Editing
- Real-Time PCR
- Microbial Efflux Pumps
- Oral Microbial Ecology
- Real-Time PCR in Food Science
- Bacterial Gene Regulation and Transcriptional Networks