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

Genome Biology, 2010

Shengdong Ke and Lawrence A. Chasin‡
Department of Biological Sciences
Columbia University
New York, NY 10027 USA
‡ To whom correspondence should be addressed:
Email: lac2@columbia.edu

Abstract
Background
A 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.
Results
Strongly 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 nt 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 (SNP) 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 hnRNPs A1/A2, C, D, F/H, G, K, L, M, PTB and for 9G8. 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.
Conclusions
The 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.

Distribution of pentamer pairs around constitutive or alternative exons.  

Left: Two intronic 50 nt regions chosen on each side of an exon generate four possible pairings. Ud, upstream distal; Up, upstream proximal; Dp, downstream proximal; Dd, downstream distal.

Links to text files containing a complete listing of 1,048,576 pentamer pairs in the UpDp, UpDd, UdDp and UdDd regions of constitutive and alternative exons and their counts and their p values are presented below:

Constitutive exons:  UpDp,  UpDd,  UdDp  and  UdDd

Alternative exons:     UpDp,  UpDd,  UdDp  and  UdDd

Note: In the listings, a label =1 denotes a positively correlation and 0 denotes a negative correlation. 

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