Short-Interfering-RNA-Mediated Gene Silencing in Mammalian Cells Requires Dicer and eIF2C Translation Initiation Factors

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.

[1]  Andrew Fire,et al.  The rde-1 Gene, RNA Interference, and Transposon Silencing in C. elegans , 1999, Cell.

[2]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[3]  K. Ui-Tei,et al.  Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target , 2000, FEBS letters.

[4]  S. Tutton,et al.  Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells , 2001, Molecular and Cellular Biology.

[5]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[6]  M. von Knebel Doeberitz,et al.  Human eukaryotic initiation factor EIF2C1 gene: cDNA sequence, genomic organization, localization to chromosomal bands 1p34-p35, and expression. , 1999, Genomics.

[7]  Magdalena Zernicka-Goetz,et al.  Specific interference with gene function by double-stranded RNA in early mouse development , 2000, Nature Cell Biology.

[8]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[9]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[10]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[11]  W. Filipowicz,et al.  Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

[13]  Patrick J. Paddison,et al.  Stable suppression of gene expression by RNAi in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Z. Zhang,et al.  Molecular cloning and characterization of a rabbit eIF2C protein. , 1998, Gene.

[15]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.

[16]  A. Pasquinelli,et al.  Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing , 2001, Cell.

[17]  T Irimura,et al.  Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase. , 2000, Biochimica et biophysica acta.

[18]  Q. Wei,et al.  RNAi as Random Degradative PCR siRNA Primers Convert mRNA into dsRNAs that Are Degraded to Generate New siRNAs , 2001, Cell.

[19]  Titia Sijen,et al.  On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing , 2001, Cell.