Conclusion
The tools and platforms are present to appropriately take into account expression data coming from splice variants. Several alternatives exist to quantify limited numbers of genes and variants. They all, however, require careful design and adequate controls to ensure that specificity is achieved. With the advent of microarray platforms to monitor and quantify splice variant expression levels, one is likely to be in a position to provide concrete answers to some of the issues that are often...
Revertant Fibres
Revertant fibres are dystrophin-positive fibres that have been detected immunohistochemically in dystrophic muscle. These dystrophin-positive fibres are found in most animal models of muscular dystrophy, and at least 50 of DMD patients Hoffman et al. 1990 Klein et al. 1992 Schatzberg et al. 1998 Sherratt et al. 1993 Wallgren-Pettersson et al. 1993 Wilton et al. 1997 . The dystrophin in revertant fibres has been shown to be internally deleted, with RNA and protein studies involving epitope...
A First Causal Therapy for SMA Patients
VPA has been shown to significantly increase SMN protein levels in fibro-blast cell lines treated with drug amounts ranging from 0.5 M to 50 M Brichta et al. 2003 . VPA is an FDA-approved drug that has been used in the therapy of epilepsy for more than three decades Zaccara et al. 1988 . More recently, VPA has also gained importance as anticonvulsant in manic depression, migraine, and dementia Papatheodorou et al. 1995 Mathew et al. 2000 Lonergan et al. 2004 . Although it is known that VPA up-...
Modulation of Splicing by Aclarubicin
Increased retention of SMN2 exon 7 has also been observed in type 1 SMA fibroblasts treated with the antibiotic aclarubicin Andreassi et al. 2001 . Anthracycline antibiotics such as aclarubicin and doxorubicin are widely used in chemotherapy against solid tumors and leukemia. Anthracyclines have also been shown to be potent differentiation inducers when used at subtoxic concentrations Chenais et al. 2000 . In this pathway, aclarubicin seems to specifically increase the expression of...
Unforeseen Modulation of Alternative Splicing by Various Molecules
For almost two decades alterations of alternative splicing have been observed for many different genes in response to various chemical compounds and hormones. Indeed, sodium butyrate was shown to modulate the splicing pattern of the calcitonin gene in human thyroid carcinoma cells Nakagawa et al. 1988 while ethanol and DMSO treatment changed the expression profile of the calcium channel subunits- and the neural cell adhesion molecule NCAM -encod-ing splice variants, respectively Walter et al....
References Ncf
Amack JD. Mahadevan MS 2001 The myotonic dystrophy expanded CUG repeat tract is necessary but not sufficient to disrupt C2C12 myoblast differentiation. Hum Mol Genet 10 1879-1887 Amack JD, Paguio AP, Mahadevan MS 1999 Cis and trans effects of the myotonic dystrophy DM mutation in a cell culture model. Hum Mol Genet 8 1975-1984 Amack JD, Reagan SR, Mahadevan MS 2002 Mutant DMPK 3'-UTR transcripts disrupt C2C12 myogenic differentiation by compromising MyoD. J Cell Biol 159 419-429 Anant S,...
The 384910 kb CT Mutation
The 3849 10 kb C T is a nucleotide substitution, 10 kb downstream nucleotide 3849, the last nucleotide of CFTR exon 19 Fig. 1a . This substitution generates a cryptic donor splice site that leads to partial inclusion of 84 bp exon between exon 19 and exon 20 Highsmith et al. 1994 . The 84 bp exon contains an in-frame stop codon and therefore the aberrantly spliced transcripts lead to the generation of a truncated protein. The 3849 10 kb C T is the twelfth most common mutation worldwide with...
Influence of SMN2 Copy Number on the SMA Phenotype
Despite apparently similar mutations, e.g. homozygous absence of SMN1, SMA patients present considerably different severities of the disease. The main cause for this fact is the variable number of SMN2 copies which directly correlates with the disease severity Burghes 1997 Brahe 2000 Feldkotter et al. 2002 . According to this observation, the majority of type I SMA patients carry two SMN2 copies, type II SMA patients three SMN2 copies, type III SMA patients three or four copies, and type IV...
Splicing Regulation of SMN Exon 7
SMN exon 7 spans 54 nt and harbors a stop codon at nt position 49 to 51. The last position of exon 7 is an adenosine residue, which places exon 7 into the minor group of internal exons lacking a 3'-end G residue Burge et al. 1999 . Exon 7 is characterized by a weak 3'splice site due to a suboptimal polypyrimidine tract Lim and Hertel 2001 . The C-to-T transition at position 6 of exon 7 840C T is the only difference between the two SMN genes localized within the coding region. However, it is a...
Splicing Modulation of CFTR Minigenes Carrying Splicing Mutations
As the first step in understanding the role of splicing regulation as a modifier of the CF disease, the effect of overexpression of splicing factors on the level of correctly spliced CFTR transcripts was studied in minigenes carrying CFTR splicing mutations Aznarez et al. 2003 Nissim-Rafinia et al. 2000 Pagani et al. 2000 Pagani et al. 2003a Pagani et al. 2003b . The studied mutations included the intronic 3849 10 kb C T and IVS8-5T and the exonic mutations in exons 9, 12, and 13, as mentioned...
Splicing Modulation of Endogenous CFTR Allele Carrying 3849 10 Kb CT Splicing
In order to investigate whether higher levels of correctly spliced transcripts can restore the CFTR function, cells comprising the entire CFTR coding region carrying a splicing mutation were studied Nissim-Rafinia et al. 2004 . An epithelial cell-line CFP15a from a nasal polyp of a CF patient, carrying the 3849 10 kb C T splicing mutation was established to analyze the effect of overexpression of splicing factors on the splicing pattern and protein function. Overexpression of Htra2-P1, SC35,...
Histone Deacetylase HDAC Inhibitors that Increase the SMNRNAProtein
One of the most exciting findings in SMA research was the identification of HDAC inhibitors as a group of drugs that increase the SMN protein levels in vitro and in vivo by activating the transcription and or correcting the splicing of SMN2. Activation and repression of gene transcription largely depends on chromatin structure. Chromatin consists of DNA, histones, and non-histone proteins. Approximately two superhelical turns of DNA containing 146 base pairs are wrapped around an octamere of...
References Sxc
Andreassi C, Jarecki J, Zhou J, Coovert DD, Monani UR, Chen X, Whitney M, Pollok B, Zhang M, Androphy E, Burghes AH 2001 Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum Mol Genet 10 2841-2849 Andreassi C, Angelozzi C, Tiziano FD, Vitali T, De Vincenzi E, Boninsegna A, Villanova M, Bertini E, Pini A, Neri G, Brahe C 2004 Phenylbutyrate increases SMN expression in vitro relevance for treatment of spinal muscular atrophy. Eur J Hum Genet...
f
Fw Donor probe Acceptor probe Fig. 3A-C. Real-Time Quantitative PCR. A Boundary-spanning primer. B Boundary-spanning Taqman probe. C Boundary-spanning hybridization probe. Only the short isoform is represented. Fw Forward Rv Reverse splicing variants of the calpain 3 gene using a junction-spanning primer. This problem was solved by using different chemistries with boundary-spanning molecular beacon and scorpion probes and adjusting the amount of reagents Taveau et al. 2002 . Molecular beacons...
Modulation of Kinase Activity
The first evidence that chemical compounds targeting enzymes involved in the phosphorylation of the SR proteins' RS domain could constitute precious tools for interfering with alternative splicing regulation came from the study of a glycosylated indolocarbazole derivative NB-506 , a potent inhibitor of DNA topoisomerase I Pilch et al. 2001 Soret and Tazi 2003 . DNA topoisomerase I is a nuclear target of a number of anticancer agents derived from the plant alkaloid camptothecin Pommier et al....