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Many diseases affect pre-mRNA splicing, and alternative splicing is a major source of proteome diversity and an important mechanism for modulating gene expression. The ability to regulate a specific splicing event is therefore desirable; for example, to understand splicing-associated pathologies. We have developed methods based on modified U7 snRNAs, which allow us to induce efficient skipping or inclusion of selected exons. Here, we have adapted these U7 tools to a regulatable system that relies on a doxycycline-sensitive version of the Krüppel-associated box (KRAB)/KAP1 transcriptional silencing. Co-transduction of target cells with two lentiviral vectors, one carrying the KRAB protein and the other the regulatable U7 cassette, allows a tight regulation of the modified U7 snRNA. No leakage is observed in the repressed state, whereas full induction can be obtained with doxycycline in ng ml−1 concentrations. Only a few days are necessary for a full switch, and the induction/repression can be repeated over several cycles without noticeable loss of control. Importantly, the U7 expression correlates with splicing correction, as shown for the skipping of an aberrant β-globin exon created by a thalassaemic mutation and the promotion of exon 7 inclusion in the human SMN2 gene, an important therapeutic target for spinal muscular atrophy.Gene Therapy (2009) 16, 70–77; doi:10.1038/gt.2008.138; published online 14 August 2008 [ABSTRACT FROM AUTHOR]

The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3' end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy. [ABSTRACT FROM AUTHOR]

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs. [ABSTRACT FROM AUTHOR]

Infection of HeLa-cell monolayer cultures with rabbit poxvirus induces a marked decrease in cell-associated protein and in the activities of lactate dehydrogenase, acid phosphatase, and β-glucuronidase. This effect begins to occur around 10 hours post-infection (p.i.) and is accompanied by a concomitant rise of these enzyme activities in the culture medium. Only few cells detach from infected monolayers and these cannot account for protein release. Virion release can be inhibited at 4° C, whereas protein release cannot and it seems therefore that these events do not happen by a common mechanism. Moreover, penetration studies with [C]-sucrose indicate that protein release reflects a true increase in plasma membrane permeability. Using the Gomori stain for acid phosphatase, a release of the enzyme into the cytoplasm around 8 hours p.i. can be confirmed rendering a causative role of lysosomal hydrolases in the pathogenesis of the observed plasma membrane permeability changes possible but not proving it. [ABSTRACT FROM AUTHOR]