Loss of Function of Surrounding Genes

Expanded CTG repeats were shown to alter chromatin structure and have regional effects on gene expression (Otten and Tapscott 1995 Wang et al. 1994). The CTG expansion in the DMPK 3'UTR are located immediately upstream of the SIX5 promoter region and were shown to lower SIX5 expression (Gennarelli et al. 1999 Inukai et al. 2000 Klesert et al. 1997 Thornton et al. 1997). Six5 is a transcription factor required for eye development in Drosophila, and the mouse homologue is implicated in distal...

Drugs that Increase the SMN Protein Level

Since demonstration that each SMA patient lacking SMN1 carries i to 4 or sometimes even more SMN2 gene copies (Burghes 1997 Brahe 2000 Feldkotter et al. 2002 Wirth et al. 2006), researchers worldwide have eagerly been searching for substances that increase SMN2--derived SMN protein levels in order to identify candidate drugs for SMA therapy. Several compounds have been described to increase SMN protein levels in fibroblasts and or lym-phoblastoid cell lines derived from SMA patients, including...

Low Medium Throughput Techniques

Until the introduction of PCR, the quantitative analysis of alternative transcripts was limited to technologies such as northern blotting or ribonuclease protection assays, both having severe limitations. Northern blotting is very time-consuming, requires a large amount of RNA, is only suitable for highly expressed transcripts, and may not be able to differentiate close isoforms (Streuli et al. 1987). RNAse protection assay is also a heavy technique, more efficient to monitor small sequence...

References

Amaral MD, Pacheco P, Beck S, Farinha CM, Penque D, Nogueira P, Barreto C, Lopes B, Casals T, Dapena J, Gartner S, Vasquez C, Perez-Frias J, Olveira C, Cabanas R, Estivill X, Tzetis M, Kanavakis E, Doudounakis S, Dork T, Tummler B, Girodon-Boulandet E, Cazeneuve C, Goossens M, Blayau M, Verlingue C, Vieira I, Ferec C, Claustres M, des Georges M, Clavel C, Birembaut P, Hubert D, Bienvenu T, Adoun M, Chomel JC, De Boeck K, Cuppens H, Lavinha J (2001) Cystic fibrosis patients with the 3272-26A>...

Sequestration of MBNL Proteins

The three human MBNL paralogues are homologues of Drosophila mus-cleblind (mbl), which is required for Drosophila photoreceptor and muscle differentiation (Artero et al. 1998 Begemann et al. 1997). MBNL1, MBNL2, and MBNL3 are located on chromosomes 3, 13, and X, respectively (Fardaei et al. 2002 Miller et al. 2000). MBNL1 was identified based on its ability to bind double-stranded CUG-repeat RNA in HeLa cell nuclear extracts (Miller et al. 2000). All three MBNL proteins colocalize with expanded...

Progress in Molecular and Subcellular Biology

M ller (Managing Editor), Ph. Jeanteur, Y. Kuchino, A. Macieira-Coelho, R.E. Rhoads Progress in Molecular and Subcellular Biology Protein Degradation in Health and Disease Regulation of Alternative Splicing Ph. Jeanteur (Ed.) Guidance Cues in the Developing Brain RNA Trafficking and Nuclear Structure Dynamics Developmental Biology of Neoplastic Growth Subseries Marine Molecular Biotechnology Volume 37 Sponges (Porifera) WE.G. M ller (Ed.)

Alternative Splicing Therapeutic Target and Tool

Alternative splicing swells the coding capacity of the human genome, expanding the pharmacoproteome, the proteome that provides targets for therapy. Splicing, both constitutive and regulated forms, can itself be targeted by conventional and molecular therapies. This review focuses on splicing as a therapeutic target with a particular emphasis on molecular approaches. The review looks at the use of antisense oligonucleotides, which can be employed to promote skipping of constitutive...

SMN the SMA Determining Gene

In 1995, Lefebvre and colleagues identified the survival of motor neuron gene 1 (SMN1) as the SMA disease-determining gene (Lefebvre et al. 1995). The SMN gene exists in two copies, SMN1 and SMN2, which are almost identical except for five nucleotide differences one each in exon 7 (840C T, codon 280, nt position 27141), exon 8 (nt position 27869) and intron 6 (nt position 27092) and another two in intron 7 (nt positions 27289 and 27404), respectively (Fig. 1 Lefebvre et al. 1995 Burglen et al....

Misregulation of Alternative Splicing

Alternative splicing is a process by which multiple mRNA isoforms are generated from individual genes. The majority of human genes undergo alternative splicing explaining, in part, the disparity between the relatively small number of genes and the complexity of the human proteome (Modrek and Lee 2002 Xu et al. 2002). Alternative splicing gives rise to protein isoforms that significantly differ in their functions (Black 2003). Alternative splicing is often regulated according to cell type or...

Exon Specific RNA Interference

A variation on the theme of short oligonucleotides that can alter ratios of splicing isoforms is exon-specific RNA interference (RNAi). Because RNAi and its clinical potential have been extensively reviewed (Dykxhoorn, Novina et al. 2003 Hommel, Sears et al. 2003 Wall and Shi 2003 Stevenson 2004 Shankar, Manjunath et al. 2005), these matters will not be described here. Suffice it to say that given the specificity of RNAi and the possibility of harnessing it to knockdown-specific mRNA isoforms...

Exonic Point Mutations Leading to Aberrant Splicing

The identification of exonic splicing motifs revealed that mutations in coding regions might also affect the splicing pattern of their pre-mRNAs. Disruption of these splicing motifs, whether by nonsense, missense, or silent mutations, might promote skipping of an exon, or of part of an exon. This was shown for several human disease genes, such as ataxia-telangiectasia mutated (ATM), neurofibromatosis (NF)-1, breast cancer (BRCA)1, survival motor neuron (SMN), and CFTR (Aznarez et al. 2003...

Clinical Trials

Exploitation of synthetic antisense oligonucleotides to alter gene transcripts is in its infancy and cannot be compared to the elegant and highly specific alternative splicing that occurs naturally. Much of the early antisense work on redirecting splicing occurred in cell-free systems, however progress in some areas has been so rapid that clinical trials to address dystrophin mutations are being planned Muntoni et al. 2005 . Several issues must be considered. Rather than one therapeutic...

Importance of Alternative Splicing for Gene Regulation

The sequencing of various eukaryotic genomes has demonstrated that a surprisingly small number of genes generate a complex proteome. For example, the estimated 20,000-25,000 human protein-coding genes give rise to 100,000-150,000 mRNA variants as estimated by EST comparison. Array analysis shows that 74 of all human genes are alternatively spliced Johnson et al. 2003 and a detailed array-based analysis of chromosome 22 and 21 suggests that every protein-coding gene could undergo alternative...

Hutchinson Gilford Progeria Syndrome

The Progeria syndrome, first described at the end of the 19th century by Jonathan Hutchinson Hutchinson 1886 and later by Hastings Gilford Gilford 1904 , is a very rare and severe developmental disorder its prevalence is about 1 in 4 to 8 million characterized by the precocious appearance of pathologies which are typical of advanced age. The median age at diagnosis is 2 years. The clinical phenotype is characterized by an overall severe growth retardation median final height 100-110 cm median...

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...

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....

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

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 Mutations and Restoration of the CFTR

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...

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....