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1.  A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia 
Human mutation  2011;32(7):773-782.
Many mutations in the skeletal-muscle sodium-channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT-AC type II intron. This is a rare class of introns that, despite having AT-AC boundaries, are spliced by the major or U2-type spliceosome. The patient's skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5′ splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2-type 5′ splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35-amino-acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patient's myotonic symptoms. This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect.
doi:10.1002/humu.21501
PMCID: PMC4109284  PMID: 21412952
skeletal muscle; myotonia; splicing; gain-of-function; simulation; channelopathy
2.  Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons 
Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, exonic, rather than intronic, cis-elements are key determinants of PK-M splicing isoform ratios. Using a systematic sub-exonic duplication approach, we identify a potent exonic splicing enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative splicing.
doi:10.1093/jmcb/mjr030
PMCID: PMC3493165  PMID: 22044881
alternative splicing; cancer metabolism; pyruvate kinase; SRSF3
3.  SpliceTrap: a method to quantify alternative splicing under single cellular conditions 
Bioinformatics  2011;27(21):3010-3016.
Motivation: Alternative splicing (AS) is a pre-mRNA maturation process leading to the expression of multiple mRNA variants from the same primary transcript. More than 90% of human genes are expressed via AS. Therefore, quantifying the inclusion level of every exon is crucial for generating accurate transcriptomic maps and studying the regulation of AS.
Results: Here we introduce SpliceTrap, a method to quantify exon inclusion levels using paired-end RNA-seq data. Unlike other tools, which focus on full-length transcript isoforms, SpliceTrap approaches the expression-level estimation of each exon as an independent Bayesian inference problem. In addition, SpliceTrap can identify major classes of alternative splicing events under a single cellular condition, without requiring a background set of reads to estimate relative splicing changes. We tested SpliceTrap both by simulation and real data analysis, and compared it to state-of-the-art tools for transcript quantification. SpliceTrap demonstrated improved accuracy, robustness and reliability in quantifying exon-inclusion ratios.
Conclusions: SpliceTrap is a useful tool to study alternative splicing regulation, especially for accurate quantification of local exon-inclusion ratios from RNA-seq data.
Availability and Implementation: SpliceTrap can be implemented online through the CSH Galaxy server http://cancan.cshl.edu/splicetrap and is also available for download and installation at http://rulai.cshl.edu/splicetrap/.
Contact: michael.zhang@utdallas.edu
Supplementary Information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr508
PMCID: PMC3198574  PMID: 21896509
5.  Antisense Oligonucleotides Delivered to the Mouse CNS Ameliorate Symptoms of Severe Spinal Muscular Atrophy 
Science translational medicine  2011;3(72):72ra18.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2′-O-2-methoxyethyl–modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system–directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.
doi:10.1126/scitranslmed.3001777
PMCID: PMC3140425  PMID: 21368223

Results 1-5 (5)