Overview of 5′ splice site (5′ ss) SNP analysis and RT–PCR assay of seven HapMap LCLs.

The effect of a given 5′ splice site SNP on splicing depends on its local sequence context. (A) Schematic diagram of the pI-11-H3 minigene splicing reporter and minigene analysis of the 5′ splice site polymorphism in CAST. (B) The maximum and minimum 5′ splice site scores of ‘splicing unaffected' exons and ‘splicing affected' exons. (C) Minigene analysis of TRIM62 and HMSD indicates that the identical set of 5′ splice site mutations has dramatically different impacts on splicing within two different exonic/intronic sequence contexts. In this experiment, we swapped in a series of 5′ splice site 9-mers (three exonic nucleotides and six intronic nucleotides) into the basic constructs of TRIM62 and HMSD, with a gradient of eight different splice site scores between 11.00 and 4.44. In all gel pictures, the number below each lane represents the percent exon inclusion level estimated by the fluorescently labeled RT–PCR. Asterisk denotes PCR products of unexpected sizes resulting from the usage of cryptic splice sites as confirmed by sequencing. 5′ ss, 5′ splice site.

The upstream 3′ splice site and downstream intronic poly-G runs function redundantly to protect a TRIM62 exon from its 5′ splice site polymorphism. (A) Trinucleotide motifs enriched within the 100 bp intronic region downstream of the ‘splicing unaffected' exons as compared with the ‘splicing affected' exons. X-axis shows motif density in ‘splicing affected' exons. Y-axis shows motif density in ‘splicing unaffected' exons. The size of each dot is proportional to the negative log of the P-value for motif enrichment. The nucleotide sequences of motifs with Bonferroni-corrected P-values of <0.05 are shown. (B) The nucleotide sequence of the TRIM62 exon and its flanking intronic regions. Box frame indicates the exon. Ten poly-G runs in the 100 bp downstream intron are underlined and denoted as G1-G10. Filled diamonds represent the positions where single-nucleotide mutations are introduced to disrupt individual poly-G runs. (C) Schematic diagrams of mutant minigene constructs with serial disruptions of poly-G runs. (D) Results of TRIM62 minigene experiments by site-directed G-to-C disruptions of poly-G runs. Disruptions of poly-G runs in the minigene constructs containing the wild-type 3′ splice site have no impact on exon splicing (left panel). Disruptions of poly-G runs in the minigene constructs containing the mutant (weakened) 3′ splice site significantly reduce the exon inclusion level of the TRIM62 exon. In the ‘Intron Deletion' constructs, only the first 6 intronic nucleotides within the 5′ splice site of the TRIM62 exon are kept when inserted into the basic pI-11-H3 minigene vector. The vector sequence that now serves as the intronic sequence immediately downstream of the TRIM62 5′ splice site contains basic splicing signals, i.e. a strong branch point and the 3′ splice site for the downstream constitutive exon. In all gel pictures, the number below each lane represents the percent exon inclusion level estimated by the fluorescently labeled RT–PCR. Asterisk denotes PCR products of unexpected sizes resulting from the usage of cryptic splice sites as confirmed by sequencing. 5′ ss, 5′ splice site; 3′ ss, 3′ splice site.

Examples of 5′ splice site SNPs causing splicing differences among seven HapMap LCLs. The genotypes of seven individuals and the estimated exon inclusion levels are indicated below each gel picture. The exon inclusion level is calculated from the fluorescently labeled RT–PCR as the intensity of the exon inclusion band(s) over the total intensity of all exon inclusion and skipping bands. (A) HMSD, (B) WDR67, (C) PLD2, (D) RCC2.

Strong upstream 3′ splice sites buffer 5′ splice site polymorphisms. (A) Box plots of splicing signals (ESE/ESS density, 3′ splice site score) of ‘splicing unaffected' exons and ‘splicing affected' exons. (B) Correlation between 3′ splice site score and 5′ splice site SNP density in 12 862 human constitutive exons. (C) The 3′ splice site strength of a CAST exon affects the impact of its 5′ splice site polymorphism on splicing. (D) The 3′ splice site strength of a TRIM62 exon affects the impact of its 5′ splice site polymorphism on splicing. In all gel pictures, the number below each lane represents the percent exon inclusion level estimated by the fluorescently labeled RT–PCR. Asterisk denotes PCR products of unexpected sizes resulting from the usage of cryptic splice sites as confirmed by sequencing. 5′ ss, 5′ splice site; 3′ ss, 3′ splice site.