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
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
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Status: |
Published |
Creators/Authors: |
Creators | Email | Pitt Username | ORCID |
---|
Levinson, N | | | | Hinman, R | | | | Patil, A | | | | Stephenson, CRJ | | | | Werner, S | | | | Woo, GHC | | | | Xiao, J | | | | Wipf, P | pwipf@pitt.edu | PWIPF | | Lynch, KW | | | |
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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: |
26 Sep 2022 16:53 |
URI: |
http://d-scholarship.pitt.edu/id/eprint/19905 |
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