Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.

Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

[1]  J. Downward,et al.  The Serine Protease Omi/HtrA2 Regulates Apoptosis by Binding XIAP through a Reaper-like Motif* , 2002, The Journal of Biological Chemistry.

[2]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[3]  K. Nishikura,et al.  A Short Primer on RNAi RNA-Directed RNA Polymerase Acts as a Key Catalyst , 2001, Cell.

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

[5]  Martin Tabler,et al.  Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila , 2001, Current Biology.

[6]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

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

[8]  A. Djikeng,et al.  RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs. , 2001, RNA.

[9]  N. Sonenberg,et al.  The major mRNA‐associated protein YB‐1 is a potent 5′ cap‐dependent mRNA stabilizer , 2001, The EMBO journal.

[10]  Phillip D. Zamore,et al.  RNA interference: listening to the sound of silence , 2001, Nature Structural Biology.

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

[12]  A. Fire,et al.  Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Pasquinelli,et al.  A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA , 2001, Science.

[14]  M Mochii,et al.  Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Jay Z. Parrish,et al.  Mitochondrial endonuclease G is important for apoptosis in C. elegans , 2001, Nature.

[16]  R. Schnabel,et al.  Essential roles for four cytoplasmic intermediate filament proteins in Caenorhabditis elegans development , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Vaucheret,et al.  RNA Silencing in Plants--Defense and Counterdefense , 2001, Science.

[18]  Ming-Bo Wang,et al.  Gene silencing as an adaptive defence against viruses , 2001, Nature.

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

[20]  T. Tuschl,et al.  RNA Interference and Small Interfering RNAs , 2001, Chembiochem : a European journal of chemical biology.

[21]  S. Peltz,et al.  A novel mRNA‐decapping activity in HeLa cytoplasmic extracts is regulated by AU‐rich elements , 2001, The EMBO journal.

[22]  P. Sharp,et al.  RNA interference--2001. , 2001, Genes & development.

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

[24]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[25]  Sebastian A. Leidel,et al.  Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III , 2000, Nature.

[26]  P. Zipperlen,et al.  Functional genomic analysis of C. elegans chromosome I by systematic RNA interference , 2000, Nature.

[27]  H. Prydz,et al.  Characterization of PSKH1, a novel human protein serine kinase with centrosomal, golgi, and nuclear localization. , 2000, Genomics.

[28]  H. Cerutti,et al.  Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. , 2000, Science.

[29]  A. Fire,et al.  Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. , 2000, Molecular cell.

[30]  H. Prydz,et al.  Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes. , 2000, Nucleic acids research.

[31]  A. Fire,et al.  dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. , 2000, Gene.

[32]  J. C. Clemens,et al.  Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  M. Wiiger,et al.  Effects of binding of ligand (FVIIa) to induced tissue factor in human endothelial cells. , 2000, Thrombosis research.

[34]  J. Cáceres,et al.  Functional characterization of SR and SR‐related genes in Caenorhabditis elegans , 2000, The EMBO journal.

[35]  C. Mello,et al.  Genetic requirements for inheritance of RNAi in C. elegans. , 2000, Science.

[36]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[37]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.

[38]  R. Lehmann,et al.  Targeted mRNA degradation by double-stranded RNA in vitro. , 1999, Genes & development.

[39]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[40]  R. Plasterk,et al.  mut-7 of C. elegans, Required for Transposon Silencing and RNA Interference, Is a Homolog of Werner Syndrome Helicase and RNaseD , 1999, Cell.

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

[42]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[43]  Michael Zuker,et al.  Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide , 1999 .

[44]  R. Carthew,et al.  Use of dsRNA-Mediated Genetic Interference to Demonstrate that frizzled and frizzled 2 Act in the Wingless Pathway , 1998, Cell.

[45]  G. Stark,et al.  How cells respond to interferons. , 1998, Annual review of biochemistry.

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

[47]  A. Kolstø,et al.  Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells. , 1996, Blood.

[48]  A. Kolstø,et al.  Cell biology of tissue factor, the principal initiator of blood coagulation. , 1996, Thrombosis research.

[49]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[50]  H. Prydz,et al.  Studies on tissue thromboplastin: I. Solubilization with sodium deoxycholate , 1966 .

[51]  H. Jensen,et al.  STUDIES ON TISSUE THROMBOPLASTIN , 1954 .