Intronic motif pairs cooperate across exons to promote pre-mRNA splicing

Shengdong Ke,Lawrence A Chasin

doi:10.1186/gb-2010-11-8-r84

Abstract

BackgroundA very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions.ResultsStrongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific.ConclusionsThe exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition.

Highlights

A very early step in splice site recognition is exon definition, a process that is as yet poorly understood
We defined four 50-nucleotide stretches in which to search for co-occurring motifs: intronic regions from -100 to -51 nucleotides (Ud, upstream distal), from -64 to -15 nucleotides (Up, upstream proximal), from +7 to +56 nucleotides (Dp, downstream proximal), and from +51 to +100 nucleotides (Dd, downstream distal)
We found that genes that have alternative exons with the motif pair TGGGG: CTGGG in the UpDp intronic regions were significantly enriched among genes preferentially expressed in prefrontal cortex, thyroid and many immune tissues (BDCA4+ dendritic cells, CD14+ monocytes, CD4+ T cells, and so on), and significantly depleted in appendix, superior cervical ganglion and skeletal muscle

Summary

Introduction

A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. All pre-mRNA splicing reactions involve the removal of an intron from between two exons and so require the pairing of the splice sites at the two ends of the intron; such pairing can be considered as a mandatory ‘intron definition’ step in splicing. It is likely that the initial recognition of most splice sites involves ‘exon definition,’ the identification of two splice sites across an exon. This idea was first put forth to explain the observation that appending a 5′ splice site downstream of the second exon in a two-exon pre-mRNA molecule greatly enhances splicing of the upstream intron in vitro [1]. Despite the wide acceptance of this idea, especially in metazoans where intron size is much greater than exon size, most biochemical investigations of splicing have focused on protein-protein interaction across introns, rather than on complexes that form across exons [3,4]

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