Pre-mRNA Secondary Structures Influence Exon Recognition

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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. Almost all human protein-coding genes contain several exons and introns. Prior to translation, introns have to be removed and exons have to be joined, which happens in a processing step called splicing that generates the mature mRNA. For most genes, certain exons can be either included or excluded from the mature mRNA. It is currently not fully understood which signals are needed to accurately recognize the boundaries of exons in the intron-containing primary transcript. As in transcriptional regulation, enhancer and silencer sequence motifs are crucial for the correct recognition of exons. Splicing regulatory proteins identify these motifs in a sequence-specific manner. In general, these proteins bind to single-stranded RNA. Here, we analyzed local secondary structures of primary transcripts and found that known splicing motifs are preferentially located in a single-stranded context. Experimental tests demonstrated that motifs in single-stranded contexts have a stronger effect on splice site selection than those located in double-stranded regions. These results help to understand the action of human mutations that change the splicing pattern and indicate that local pre-mRNA secondary structures influence exon recognition.