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Use of transcriptional synergy to augment sensitivity of a splicing reporter assay

Levinson, N and Hinman, R and Patil, A and Stephenson, CRJ and Werner, S and Woo, GHC and Xiao, J and Wipf, P and Lynch, KW (2006) Use of transcriptional synergy to augment sensitivity of a splicing reporter assay. RNA, 12 (5). 925 - 930. ISSN 1355-8382

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Abstract

A primary limitation in the development and use of screens to identify factors that regulate mammalian pre-mRNA splicing has been the development of sensitive reporter assays. Alternative splicing typically involves relatively small (< 10-fold) changes in isoform ratios. Therefore, reporter constructs designed to allow direct analysis of isoform expression historically have at most a 10-fold window of discrimination between a positive signal and background. Here we describe the design and application of a reporter cell line that makes use of the phenomenon of transcriptional synergy to amplify the detection of changes in splicing, such that a three- to five-fold change in splicing pattern is observed as a 30- to 50-fold change in GFP expression. Using this cell line we have identified two small molecules, from a library of approximately 300 synthetic compounds, that can induce partial repression of a variable exon from the CD45 gene. We propose that the concept of transcription-based amplification of signal will allow the development of true high-throughput screening approaches to identify effectors of mammalian alternative splicing. Copyright © 2006 RNA Society.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Levinson, N
Hinman, R
Patil, A
Stephenson, CRJ
Werner, S
Woo, GHC
Xiao, J
Wipf, Ppwipf@pitt.eduPWIPF
Lynch, KW
Date: 1 May 2006
Date Type: Publication
Journal or Publication Title: RNA
Volume: 12
Number: 5
Page Range: 925 - 930
DOI or Unique Handle: 10.1261/rna.8306
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Refereed: Yes
ISSN: 1355-8382
MeSH Headings: Alternative Splicing; Antigens, CD45--drug effects; Antigens, CD45--genetics; Cell Line; Exons; Gene Library; Genes, Reporter; Green Fluorescent Proteins--metabolism; Luminescent Proteins--drug effects; Luminescent Proteins--genetics; Lymphocyte Activation--drug effects; RNA Precursors--metabolism; RNA Splicing; Sensitivity and Specificity; Signal Transduction; T-Lymphocytes--cytology; T-Lymphocytes--drug effects; T-Lymphocytes--metabolism; Tetradecanoylphorbol Acetate--pharmacology; Transcription, Genetic; Transfection
Other ID: NLM PMC1440914
PubMed Central ID: PMC1440914
PubMed ID: 16556940
Date Deposited: 30 Oct 2013 16:59
Last Modified: 02 Feb 2019 15:56
URI: http://d-scholarship.pitt.edu/id/eprint/19905

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