Skip Navigation



Human Molecular Genetics Advance Access published online on December 15, 2005
Human Molecular Genetics, doi:10.1093/hmg/ddi450
This Article
Right arrow Advance Access manuscript (PDF) Freely available
Right arrow All Versions of this Article:
15/2/329    most recent
ddi450v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Xu, D.-Q.
Right arrow Articles by Mattox, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, D.-Q.
Right arrow Articles by Mattox, W.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved
Received October 5, 2005
Revised December 8, 2005
Accepted December 8, 2005

Article

Identification of a splicing enhancer in MLH1 using COMPARE a new assay for determination of relative RNA splicing efficiencies

Dong-Qing Xu 1 and William Mattox 2 *

1 Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center and The Genes & Development Graduate Program, UT Graduate School of Biomedical Sciences, Houston, TX 77030
2 Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center and The Genes & Development Graduate Program, UT Graduate School of Biomedical Sciences, 1515 Holcombe Blvd, Unit 1006, Houston, TX 77030

* To whom correspondence should be addressed.
William Mattox, E-mail: wmattox{at}mdanderson.org


  Abstract

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 disease, 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 if 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 nonpolyposis 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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.