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1.  Tetracyclines That Promote SMN2 Exon 7 Splicing as Therapeutics for Spinal Muscular Atrophy 
There is at present no cure or effective therapy for spinal muscular atrophy (SMA), a neurodegenerative disease that is the leading genetic cause of infant mortality. SMA usually results from loss of the SMN1 (survival of motor neuron 1) gene, which leads to selective motor neuron degeneration. SMN2 is nearly identical to SMN1 but has a nucleotide replacement that causes exon 7 skipping, resulting in a truncated, unstable version of the SMA protein. SMN2 is present in all SMA patients, and correcting SMN2 splicing is a promising approach for SMA therapy. We identified a tetracycline-like compound, PTK-SMA1, which stimulates exon 7 splicing and increases SMN protein levels in vitro and in vivo in mice. Unlike previously identified molecules that stimulate SMN production via SMN2 promoter activation or undefined mechanisms, PTK-SMA1 is a unique therapeutic candidate in that it acts by directly stimulating splicing of exon 7. Synthetic small-molecule compounds such as PTK-SMA1 offer an alternative to antisense oligonucleotide therapies that are being developed as therapeutics for a number of disease-associated splicing defects.
PMCID: PMC2818805  PMID: 20161659
2.  Control of Pre-mRNA Splicing by the General Splicing Factors PUF60 and U2AF65 
PLoS ONE  2007;2(6):e538.
Pre-mRNA splicing is a crucial step in gene expression, and accurate recognition of splice sites is an essential part of this process. Splice sites with weak matches to the consensus sequences are common, though it is not clear how such sites are efficiently utilized. Using an in vitro splicing-complementation approach, we identified PUF60 as a factor that promotes splicing of an intron with a weak 3′ splice-site. PUF60 has homology to U2AF65, a general splicing factor that facilitates 3′ splice-site recognition at the early stages of spliceosome assembly. We demonstrate that PUF60 can functionally substitute for U2AF65 in vitro, but splicing is strongly stimulated by the presence of both proteins. Reduction of either PUF60 or U2AF65 in cells alters the splicing pattern of endogenous transcripts, consistent with the idea that regulation of PUF60 and U2AF65 levels can dictate alternative splicing patterns. Our results indicate that recognition of 3′ splice sites involves different U2AF-like molecules, and that modulation of these general splicing factors can have profound effects on splicing.
PMCID: PMC1888729  PMID: 17579712
3.  Comprehensive splice-site analysis using comparative genomics 
Nucleic Acids Research  2006;34(14):3955-3967.
We have collected over half a million splice sites from five species—Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana—and classified them into four subtypes: U2-type GT–AG and GC–AG and U12-type GT–AG and AT–AC. We have also found new examples of rare splice-site categories, such as U12-type introns without canonical borders, and U2-dependent AT–AC introns. The splice-site sequences and several tools to explore them are available on a public website (SpliceRack). For the U12-type introns, we find several features conserved across species, as well as a clustering of these introns on genes. Using the information content of the splice-site motifs, and the phylogenetic distance between them, we identify: (i) a higher degree of conservation in the exonic portion of the U2-type splice sites in more complex organisms; (ii) conservation of exonic nucleotides for U12-type splice sites; (iii) divergent evolution of C.elegans 3′ splice sites (3′ss) and (iv) distinct evolutionary histories of 5′ and 3′ss. Our study proves that the identification of broad patterns in naturally-occurring splice sites, through the analysis of genomic datasets, provides mechanistic and evolutionary insights into pre-mRNA splicing.
PMCID: PMC1557818  PMID: 16914448

Results 1-3 (3)