Pre-mRNA Secondary Structures Influence Exon Recognition

Michael Hiller,Zhaiyi Zhang,Rolf Backofen,Stefan Stamm,Yoshihide Hayashizaki

doi:10.1371/journal.pgen.0030204

Michael Hiller, Zhaiyi Zhang + Show 3 more

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https://doi.org/10.1371/journal.pgen.0030204

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Abstract

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.

Highlights

RNA molecules can adopt various conformations in solution by base pairings and hydrophobic interactions
Defined RNA structures can be recognized by other molecules, as exemplified by aminoacyl-transfer RNA (tRNA) synthetases that contact with the minor groove of the tRNA acceptor stem [3], which illustrates the functional importance of RNA structure
These results help to understand the action of human mutations that change the splicing pattern and indicate that local pre-messenger RNA (mRNA) secondary structures influence exon recognition

Summary

Introduction

RNA molecules can adopt various conformations in solution by base pairings and hydrophobic interactions. Transfer RNA (tRNA) adopts a defined secondary and tertiary structure [1], whereas most messenger RNAs (mRNAs) exhibit only local structures [2]. Defined RNA structures can be recognized by other molecules, as exemplified by aminoacyl-tRNA synthetases that contact with the minor groove of the tRNA acceptor stem [3], which illustrates the functional importance of RNA structure. Most proteins regulating splice site selection recognize singlestranded, not base-paired RNA. The sequence specificity of binding is achieved by hydrophobic interactions between RNA bases and amino acids on the surface of the protein, which explains why RNA binding proteins can bind with sequence specificity to unstructured RNA [4]

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