Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5′ splice site (5′ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5′ free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5′ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells

Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1

Ribozyme-mediated trans-splicing of a trinucleotide repeat.

Trinucleotide repeat expansions (TREs) are a recently described class of mutations characterized by a change in the size of the genomic fragment due to amplification of the repeated unit. A number of diseases have been attributed to TRE, including Huntington disease and myotonic dystrophy (DM), but attempts at genetic therapy have yet to prove successful. A potential therapeutic approach would be to repair the expanded repeat using the trans-splicing ability of group I intron ribozymes. We have used DM as a model to test this hypothesis. A group I intron ribozyme (DMPK-RZ1) was designed to modify the TRE at the 3' end of the human myotonic dystrophy protein kinase (DMPK) transcripts. DMPK-RZ1 was shown to ligate a small DMPK mRNA fragment, contained within the ribozyme, to a simple DMPK-target RNA in vitro. It also modified a larger target transcript, leading to replacement of twelve repeats with five repeats, both in vitro and in mammalian cells. Finally, this ribozyme successfully replaced the 3' end of endogenous DMPK mRNA in fibroblasts with a different 3' region. Ribozyme-mediated RNA repair may thus form a novel therapeutic strategy for diseases associated with repeat expansions