Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies.
Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within protein-coding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in diseases, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict whether a given mutation will have effects on splicing based on sequence alone. Here, we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary non-polyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659, indicating that their primary effect is on splicing. Surprisingly, the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together, our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion.
Xu
Dong-Qing
DQ
Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, and The Genes and Development Graduate Program, UT Graduate School of Biomedical Sciences, 1515 Holcombe Blvd, Unit 1006, Houston, 77030, USA.
Mattox
William
W
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000235:genetics*; D018389:Codon, Nonsense / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D017931:DNA Primers; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D000070957:MutL Protein Homolog 1; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018389:Codon, Nonsense; D017931:DNA Primers; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1