A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Summary

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Abstract

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor. However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance. We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA. The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA. A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs. Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction. This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.

Protocol

Pool design and oligo recovery The first step is to design the pre-mRNA pool to be studied. This can be done using the UCSC Genome Browser and downloading particular genes, splice junctions, or other areas of interest. Once the windows of interest have been selected, tile across them computationally using the following conditions: read length should be 30 nucleotides with a 10 nucleotide overlap, therefore each oligo is shifted 20 nucleotides from the prior one. *Oligo overlap should be increased…

Discussion

When doing this procedure it’s important to remember that creation of the pool is flexible and open to modification at either the RNA or protein level. At the RNA level orthologous regions, disease mutations, polymorphisms, or random mutations can be introduced into the oligonucleotide sequence. At the protein level the RNA binding factor can be modified by phosphorylation or other post translational modifications. Additionally, the binding environment can be manipulated to either increase or decrease the levels …