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Updated: Jun 19, 2026
11:34
Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
Published on: August 9, 2019
Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA
Daniel C Reid1, Brian L Chang, Samuel I Gunderson
1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
RNA (New York, N.Y.)
|October 29, 2009
Summary
Researchers developed a new in vitro method to map splicing factor binding sites on pre-mRNA, overcoming biases of previous techniques. This approach reveals new insights into RNA-binding protein interactions and their roles in gene regulation.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Regulation
Background:
- Splicing factors interact with both mRNA and pre-mRNA, crucial for gene expression.
- In vivo cross-linking immunoprecipitation (CLIP) identifies these interactions but suffers from sampling bias towards abundant RNAs.
- A need exists for methods that can survey binding on pre-mRNA without bias.
Purpose of the Study:
- To develop a novel in vitro approach for surveying splicing factor binding on pre-mRNA.
- To overcome the sampling bias inherent in existing in vivo methods.
- To map splicing factor binding sites and quantify the influence of secondary structure.
Main Methods:
- Designed oligonucleotide pools tiling pre-mRNA sequences.
- Partitioned pools into bound and unbound fractions.
- Quantified fractions using two-color microarrays to map binding sites.
Main Results:
- Successfully mapped splicing factor binding sites around approximately 4000 exons.
- Validated the method by confirming U1snRNP binding specificity at the 5' splice site and identifying PTB enrichment at polypyrimidine tracts.
- Identified structural determinants and multiple binding motifs for PTB, correlating with in vivo targets.
Conclusions:
- The novel in vitro method provides an unbiased approach to survey pre-mRNA binding by splicing factors.
- The study reveals specific binding patterns for U1snRNP and PTB, including sequence and structural preferences.
- This method offers a powerful tool for understanding RNA-protein interactions in pre-mRNA processing and gene regulation.
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