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High-Throughput Screening Method to Identify Alternative Splicing Regulators

Cheryl Stork1, Sika Zheng2

  • 1Division of Biomedical Sciences, University of California, Riverside, Riverside, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|April 20, 2018
PubMed

Insights

Researchers developed a novel high-throughput screening method using dual-fluorescence minigene reporters to accurately identify splicing regulators, accelerating disease research.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Misregulation of alternative pre-mRNA splicing is implicated in numerous human diseases.
  • Identifying splicing regulators, often RNA binding proteins, has been challenging and slow.
  • Existing high-throughput methods for splicing assays often lack sensitivity and specificity.

Purpose of the Study:

  • To develop a sensitive and specific high-throughput screening method for identifying exon splicing regulators.
  • To overcome limitations of current splicing assay sensitivity and specificity.

Main Methods:

  • Utilized dual-fluorescence minigene reporters for sensitive detection of exon splicing changes.
  • Employed two complementary minigenes expressing GFP and RFP oppositely for enhanced specificity.
  • Designed the method for screening cDNA, shRNA, or chemical libraries.

Main Results:

  • The dual-fluorescence reporter system allows for sensitive detection of exon splicing changes.
  • The complementary reporter design significantly reduces false positives, enhancing specificity.
  • The method enables sensitive identification of true splicing regulators.

Conclusions:

  • This novel screening method offers a sensitive and specific approach to identify splicing regulators.
  • The technique has broad applicability for discovering splicing modulators from various libraries.
  • Accelerating the discovery of splicing regulators can aid in understanding and treating diseases.
Keywords:
Alternative splicingDual-fluorescence minigene reportersGFPHigh-throughput screenRFP

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