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.

Binding of the zymogen serine protease Factor VII (FVII) to its cellular cofactor tissue factor (TF) triggers blood coagulation. Several recent reports have suggested that the formation of this complex may serve additional functions. We have used cDNA arrays to study differential gene expression in response to the interaction of activated FVII (FVIIa) with TF on a human keratinocyte cell line. Of 931 mRNA species observed up to 6 h after FVIIa (10 nM) addition, 24 were significantly up-regulated in what may resemble a wound-type response. Responders included mRNA species coding for transcription regulators (c-fos, egr-1, ETR101, BTEB2, c-myc, fra-1, and tristetraproline), growth factors (amphiregulin, hbEGF, CTGF, and FGF-5), proinflammatory cytokines (IL-1␤, IL-8, LIF, and MIP2␣), proteins involved in cellular reorganization/migration (RhoE, uPAR, and collagenases 1 and 3), and others (PAI-2, cyclophilin, GADD45, Jagged1, and prostaglandin E 2 receptor). The transcriptional response to FVIIa was abrogated by antibodies to TF and left unaffected by hirudin. The pattern of genes induced suggests that the FVIIa⅐TF complex may play an active role in early wound repair as well as hemostasis. The former is a novel function ascribed to the complex that may also be contributing to the pathophysiology of unwarranted TF expression. Tissue factor (TF)1 is a transmembrane glycoprotein that is constitutively expressed at high levels in tissues surrounding inner and outer bodily surfaces but not in cells that are in direct contact with blood (1). Disruption of these boundaries or induction of TF synthesis in monocytes (2) and endothelial cells (1) will allow plasma coagulation factors to establish contact with TF-expressing cells (1). TF then acts as a cofactor supporting activation of the zymogen serine protease FVII to FVIIa as well as the activity of FVIIa toward its substrates. Once formed, the FVIIa⅐TF complex triggers activation of zymogen coagulation proteases, which ultimately serve to cleave fibrinogen and generate a fibrin clot. Acting in concert with the accumulation of platelets, this is the main line of protection against blood loss after injury.Recent reports have shown that FVIIa can also induce TFdependent cellular signaling independent of downstream activation of the coagulation cascade (3-5). FVIIa binding to TF triggers intracellular calcium mobilization through phosphatidylinositol-specific phospholipase C (3, 4) as well as activation of mitogen-activated protein kinases (MAPKs) (5, 6) in several cell types.In this study we have addressed the physiological role of FVIIa⅐TF signaling by looking for transcriptional consequences of FVIIa-induced activation of HaCaT cells (7), a spontaneously transformed keratinocyte line much used as a keratinocyte model. These cells respond to regulatory signals in a similar manner to normal keratinocytes (8), express a range of epidermal differentiation markers (8), and are able to support epidermal differentiation and basement membrane formation in surface transplants (9)...

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.