RNA interference : OFF switch for gene expression ... RNA interference : OFF switch for gene...

RNA interference : OFF switch for gene expression ... RNA interference : OFF switch for gene expression
RNA interference : OFF switch for gene expression ... RNA interference : OFF switch for gene expression
RNA interference : OFF switch for gene expression ... RNA interference : OFF switch for gene expression
RNA interference : OFF switch for gene expression ... RNA interference : OFF switch for gene expression
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  • RNA interference : OFF switch for gene expression in eukaryotes Hiroaki Tabara Kyoto University Graduate School of Medicine, HMRO

    The central dogma of molecular biology is the flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation). In eukaryotes (organisms with a nucleus), post-transcriptional gene regulations are interesting and mysterious events. This short review focuses on post-transcriptional regulations mediated by double-stranded RNA (dsRNA). RNA interference (RNAi)

    RNAi is a form of sequence-specific gene silencing induced by the introduction of dsRNA. The ability of dsRNA to induce silencing was first discovered in the nematode C. elegans [1], and similar events have since been observed in a variety of eukaryotes. Technological aspect

    RNAi is a physiological response useful for experimental control of gene expression. The application of RNAi began at basic researches in C. elegans, Drosophila (fly), plants,

    Trypanosoma (protist), etc. In these organisms, long dsRNA (roughly > 500 base pairs) is commonly utilized for the silencing experiment. There are several methods of introducing dsRNA into organisms. In vitro-synthesized dsRNA can be introduced by micro-injection, feeding or transfection. Alternatively, scientists can make transgenic organisms that express dsRNAs from transgenes.

    By contrast, the introduction of long dsRNA causes non-specific harmful effects in mammalian cells. In order to avoid this problem, RNAi experiments in mammalian systems are now performed with small RNAs mimicing an intermediate dsRNA in RNAi [2] or precursors of micro-RNAs. Mechanism

    RNAi results in a reduction in the steady state level of the targeted endogenous mRNA. In animals, major steps in the RNAi response are thought to occur at a post-transcriptional level. The mechanisms of RNAi and related post-transcriptional gene silencing (PTGS) have been aggressively studied.

    In several model organisms including C. elegans [3], scientists screened for mutants whose RNAi/PTGS responses are defective. These genetic screens have identified some important genes required for RNAi/PTGS.

    An interesting discovery was that plants exhibiting PTGS contain small RNAs, about 25 nucleotides (nt) length, derived from the sequence of the silenced gene [4]. This small RNA appears to be a key of RNAi/PTGS. Subsequent studies in Drosophila have shown that the introduced dsRNAs are processed by a ribonuclease III activity and converted into small RNAs (small interfering RNAs; siRNA) [5, 6]. The siRNAs are incorporated into a complex termed RNA-induced silencing complex (RISC), and the RNA-protein complex is thought to recognize and destruct the target

  • mRNA [7]. Additionally, RNA-dependent RNA polymerase (RdRP) activities seem to be required for RNAi/PTGS responses in some (but not all) organisms. Physiological aspect

    Viruses and transposons (mobile elements) are parasites rich in nucleic acids. Viruses shuttles between hosts and the environment, and transposons can mobilize within each individual cell of hosts. Studies in plants have suggested that the PTGS response is similar to an anti-viral response. Somewhat similarly, a portion of the RNAi machinery appears to overlap with a mechanism of transposon silencing in C. elegans.

    There is another intersection. RNAi pathways share features with a developmental gene regulatory pathway that involves natural dsRNA encoding genes, recently named micro-RNA (miRNA) genes. Natural miRNA genes encode RNA products (about 70 nt) which are predicted to fold into stable stem-loop structures that are processed into mature miRNAs (about 22 nt). RNA-protein complexes containing the mature miRNAs are thought to inhibit translation of the target mRNA in animals or destruct the target mRNA in plants.

    Figure 1. The introduction of dsRNA causes a reduction in the level of target mRNA. Nematodes were treated with a dsRNA homologous to a muscle-expressed gene (unc-22). The expression of unc-22 mRNA was analyzed with in situ hybridization.

    References in this review (1) A. Fire, et al., Nature 391, 806-811 (1998). (2) S. M. Elbashir, et al., Nature 411, 494-498 (2001). (3) H. Tabara, et al., Cell 99, 123-132 (1999). (4) A. J. Hamilton, D. C. Baulcombe, Science 286, 950-952 (1999). (5) P. D. Zamore, et al., Cell 101, 25-33 (2000). (6) E. Bernstein, et al., Nature 409, 363-366 (2001). (7) S. M. Hammond, et al., Nature 404, 293-296 (2000).

  • Title: RNA interference (RNAi): A new mechanism by which the fragile X mental retardation protein acts in the normal brain?

    Haruhiko Siomi, Ph.D. Institute for Genome Research University of Tokushima

    Abstract:

    Fragile X syndrome is the most common familial form of mental retardation caused by

    loss-of-function mutations in the FMR1 gene. The FMR1 gene encodes an RNA-binding

    protein that associates with translating ribosomes and acts as a negative translational

    regulator. Recent work in Drosophila melanogaster has shown that the fly homolog of

    FMR1 (dFMR1) plays an important role in regulating neuronal morphology, which may

    underlie the observed deficits in behaviors of dFMR1 mutant flies. Biochemical analysis

    has revealed that dFMR1 forms a complex that includes ribosomal proteins and,

    surprisingly, Argonaute2 (AGO2), an essential component of the RNA-induced

    silencing complex (RISC) that mediates RNA interference (RNAi) in Drosophila.

    dFMR1 also associates with Dicer, another essential processing enzyme of the RNAi

    pathway. Moreover, micro-RNAs (miRNAs) can co-immunoprecipitate with dFMR1.

    Together these findings suggest that dFMR1 functions in an RNAi-related apparatus to

    regulate the expression of its target genes at the level of translation. These findings raise

    the possibility that fragile X syndrome may be the result of a protein synthesis

    abnormality caused by a defect in an RNAi-related apparatus. Recently, we have produced fly strains that lack dFMR1 or AGO2 or both. Here we present our recent results that imply an RNAi-related mechanism may be involved in the neuronal

    morphology and function. Because the core mechanisms of complex behaviors such as learning and memory, and circadian rhythms appear to be conserved, studies of fragile

    X syndrome using Drosophila as a model provide an economy-of-scale for identifying

    biological processes that likely underlie the abnormal morphology of dendritic spines

    and behavioral disturbances observed in fragile X patients.

  • References:

    Ishizuka A, Siomi MC, Siomi H. (2002): A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev. 16:2497-2508.

    Siomi MC, Higashijima K, Ishizuka A, Siomi H. (2002): Casein kinase II phosphorylates the fragile X mental retardation protein and modulates its biological properties. Mol Cell Biol. 22:8438-8447.

    Inoue SB, Shimoda M, Nishinokubi I, Siomi MC, Okamura M, Nakamura ., Kobayashi S, Ishida N, Siomi H (2002): A role for the Drosophila fragile X-related gene in circadian output. Curr. Biol. 12:1331-1335.

    Siomi, H., and Dreyfuss, G. 1997. RNA-binding proteins as regulators of gene expression. Curr. Opin. Genet. Devel. 7: 345-353.

    Siomi, M. C., Zhang, Y., Siomi, H. and Dreyfuss, G. 1996. Specific sequences in the fragile X syndrome protein FMR1 and the FXR proteins mediate their binding to 60S ribosomal subunits and the interaction among them. Mol. Cell. Biol. 16: 3825-3832.

    Siomi H, Siomi MC, Nussbaum RL, Dreyfuss G (1993): The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell 74: 291-298.

    Our recent finding that the Drosophila fragile X protein is present in the RNAi complex has been discussed in Science (Editors’ Choice 298: 497, 2002), Nature Medicine (News & Views 8:1204-1205, 2002), The Scientist (Frontlines, Oct. 14, 2002), Modern Drug Discovery (72, May 2003), and many other journals, with such words as “FMRP may play a role in RNAi, thereby implicating defects in RNAi in human disease”, “it provides the first link between RNAi and human disease” and “this finding might have serendipitously led researchers to the real cause of fragile X syndrome, a defect in RNAi activity in neurons”.

    A model of FMR1-mediated regulation of gene expression

    Haruhiko Siomi

    AAA...An7mGpppG

    translational inhibition

    dFMR1

    dFMR1

    Dmp68 AGO2

    Dmp68 AGO2

    miRNA

    Ribosome

    L5 L11