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

被引:559
作者
Holen, T [1 ]
Amarzguioui, M [1 ]
Wiiger, MT [1 ]
Babaie, E [1 ]
Prydz, H [1 ]
机构
[1] Univ Oslo, Biotechnol Ctr Oslo, N-0349 Oslo, Norway
关键词
D O I
10.1093/nar/30.8.1757
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1757 / 1766
页数:10
相关论文
共 51 条
  • [11] RNA interference is mediated by 21-and 22-nucleotide RNAs
    Elbashir, SM
    Lendeckel, W
    Tuschl, T
    [J]. GENES & DEVELOPMENT, 2001, 15 (02) : 188 - 200
  • [12] Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells
    Elbashir, SM
    Harborth, J
    Lendeckel, W
    Yalcin, A
    Weber, K
    Tuschl, T
    [J]. NATURE, 2001, 411 (6836) : 494 - 498
  • [13] Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate
    Elbashir, SM
    Martinez, J
    Patkaniowska, A
    Lendeckel, W
    Tuschl, T
    [J]. EMBO JOURNAL, 2001, 20 (23) : 6877 - 6888
  • [14] The major mRNA-associated protein YBA is a potent 5′ cap-dependent mRNA stabilizer
    Evdokimova, V
    Ruzanov, P
    Imataka, H
    Raught, B
    Svitkin, Y
    Ovchinnikov, LP
    Sonenberg, N
    [J]. EMBO JOURNAL, 2001, 20 (19) : 5491 - 5502
  • [15] Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans
    Fire, A
    Xu, SQ
    Montgomery, MK
    Kostas, SA
    Driver, SE
    Mello, CC
    [J]. NATURE, 1998, 391 (6669) : 806 - 811
  • [16] Functional genomic analysis of C-elegans chromosome I by systematic RNA interference
    Fraser, AG
    Kamath, RS
    Zipperlen, P
    Martinez-Campos, M
    Sohrmann, M
    Ahringer, J
    [J]. NATURE, 2000, 408 (6810) : 325 - 330
  • [17] A novel mRNA-decapping activity in HeLa cytoplasmic extracts is regulated by AU-rich elements
    Gao, M
    Wilusz, CJ
    Peltz, SW
    Wilusz, J
    [J]. EMBO JOURNAL, 2001, 20 (05) : 1134 - 1143
  • [18] Functional genomic analysis of cell division in C-elegans using RNAi of genes on chromosome III
    Gönczy, P
    Echeverri, C
    Oegema, K
    Coulson, A
    Jones, SJM
    Copley, RR
    Duperon, J
    Oegema, J
    Brehm, M
    Cassin, E
    Hannak, E
    Kirkham, M
    Pichler, S
    Flohrs, K
    Goessen, A
    Leidel, S
    Alleaume, AM
    Martin, C
    Özlü, N
    Bork, P
    Hyman, AA
    [J]. NATURE, 2000, 408 (6810) : 331 - 336
  • [19] Genetic requirements for inheritance of RNAi in C-elegans
    Grishok, A
    Tabara, H
    Mello, CC
    [J]. SCIENCE, 2000, 287 (5462) : 2494 - 2497
  • [20] A species of small antisense RNA in posttranscriptional gene silencing in plants
    Hamilton, AJ
    Baulcombe, DC
    [J]. SCIENCE, 1999, 286 (5441) : 950 - 952