Use of transcriptional synergy to augment sensitivity of a splicing reporter assay.
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.
Department of Biochemistry, UT Southwestern Medical Center, Dallas, Texas 75390-9038, USA
Noah
N
Levinson
Rochelle
R
Hinman
Ashwini
A
Patil
Corey R J
CR
Stephenson
Stefan
S
Werner
Grace H C
GH
Woo
Jingbo
J
Xiao
Peter
P
Wipf
Kristen W
KW
Lynch
D017398:Alternative Splicing; D002460:Cell Line; D005091:Exons; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000187:drug effects / Q000235:genetics; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics; D008213:Lymphocyte Activation / Q000187:drug effects; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
D008164:Luminescent Proteins; D012322:RNA Precursors; D049452:Green Fluorescent Proteins; D017493:Leukocyte Common Antigens; D013755:Tetradecanoylphorbol Acetate