Hybrid splicing minigene and antisense oligonucleotides as efficient tools to determine functional protein/RNA interactions

Piotr Cywoniuk,Katarzyna Taylor,Krzysztof Sobczak,Łukasz J Sznajder

doi:10.1038/s41598-017-17816-x

Piotr Cywoniuk, Katarzyna Taylor + Show 2 more

Open Access

https://doi.org/10.1038/s41598-017-17816-x

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Abstract

Alternative splicing is a complex process that provides a high diversity of proteins from a limited number of protein-coding genes. It is governed by multiple regulatory factors, including RNA-binding proteins (RBPs), that bind to specific RNA sequences embedded in a specific structure. The ability to predict RNA-binding regions recognized by RBPs using whole-transcriptome approaches can deliver a multitude of data, including false-positive hits. Therefore, validation of the global results is indispensable. Here, we report the development of an efficient and rapid approach based on a modular hybrid minigene combined with antisense oligonucleotides to enable verification of functional RBP-binding sites within intronic and exonic sequences of regulated pre-mRNA. This approach also provides valuable information regarding the regulatory properties of pre-mRNA, including the RNA secondary structure context. We also show that the developed approach can be used to effectively identify or better characterize the inhibitory properties of potential therapeutic agents for myotonic dystrophy, which is caused by sequestration of specific RBPs, known as muscleblind-like proteins, by mutated RNA with expanded CUG repeats.

Highlights

Alternative splicing (AS) is a co-transcriptional process that leads to a significant increase in proteome diversity[1,2,3]
Atp2a1 pre-mRNA contains alternative exon 22, which as confirmed by mutagenesis, is positively regulated by the binding of all three muscleblind-like proteins (MBNLs) paralogs to two YGCY motif-containing regions that are localized within intron 22, ~110 nucleotides downstream of ex[22] (Fig. 1a,c)[14,24]
We showed that short locked nucleic acid (LNA) were the least effective in vitro, most likely due to covering only a part of the motifs bound by MBNL1

Summary

Introduction

Alternative splicing (AS) is a co-transcriptional process that leads to a significant increase in proteome diversity[1,2,3]. To confirm the applicability of AONs for the verification of RBP/RNA interactions, we selected five additional pre-mRNAs, besides Atp2a1, with potential MBNL-binding motifs within an intronic sequence downstream or upstream of Pphln[1] ex6 and NASP ex[7], respectively, as well as within exonic sequences of ex[10] of Ldb[3], ex7 of Nfix and ex[1] of Mbnl[1] (Fig. 2a).

Results

Conclusion