Intronic sequences flanking alternatively spliced exons are conserved between human and mouse

Abstract

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.

Figure 1

Finding exon-skipping events that are conserved between human and mouse: 3583 exon-skipping events were found in the human genome, using the methods described in Sorek et al. (2002). (A) For 980 of these human exons, a mouse EST spanning the intron that represents the exon-skipping variant was found. Human ESTs appear in purple; mouse ESTs are in light blue. (B,C) The two possible ways to identify an exon as conserved in mouse. (B) Identification of mouse ESTs that contain the exon, as well as the two flanking exons. (C) If the exon was not represented in mouse ESTs, the sequence of the human exon was searched against the intron spanned by the skipping mouse EST on the mouse genome. If a significant conservation (above 80%) was found, and the alignment spanned the full length of the human exon, the exon was declared as conserved.

Figure 2

Per-position conservation near alternatively and constitutively spliced exons. Intronic regions near the splice site were aligned, using GAP (global alignment program) of the GCG package, and identity levels were calculated for each position as described in Methods. All 243 alternative exons and 1966 constitutive exons were used for this analysis. (A) Conservation near the 3′ splice site. Data for the last 30 nt of the intron and the first 5 nt of the exon are shown. Dashed line marks the border between the intron and the exon. (B) Conservation near the 5′ splice site. Data for the last 5 nt of the exon and the first 30 nt of the intron are shown.

Figure 2

Per-position conservation near alternatively and constitutively spliced exons. Intronic regions near the splice site were aligned, using GAP (global alignment program) of the GCG package, and identity levels were calculated for each position as described in Methods. All 243 alternative exons and 1966 constitutive exons were used for this analysis. (A) Conservation near the 3′ splice site. Data for the last 30 nt of the intron and the first 5 nt of the exon are shown. Dashed line marks the border between the intron and the exon. (B) Conservation near the 5′ splice site. Data for the last 5 nt of the exon and the first 30 nt of the intron are shown.

Figure 3

Human–mouse alignment of the KCND3 gene, corresponding to RefSeq NM_004980 (from VISTA browser, http://pipeline.lbl.gov/vistabrowser/). x-axis: The nucleotide coordinates on human chromosome 1, according to the assembly version of the human genome from June 2002. y-axis: The level of conservation between the human genome and the corresponding mouse genome, according to the MGSCv3 assembly version of the mouse genome. (A) Blue bars above the conservation area correspond to annotated exons 4–8 of KCND3. Blue areas within the conservation graph mark exons; orange areas mark conserved nonexonic sequences. The exon marked with an arrow (exon 6) is an alternatively spliced one; the others are constitutively spliced exons. (B) Enlarged view of the conservation graphs of the alternatively spliced exon (exon 6), and one of the constitutively spliced exons (exon 4) is presented to show the relative lengths of the conserved areas near the exons. (C) Human, mouse, and rat alignment of exon 6, as well as the 100 bases upstream and downstream of the exon. Exon sequence is bold; asterisks mark identity in all three organisms. Bold and underline mark the hexamer TGCATG, which previously showed the ability to regulate alternative splicing when found in introns downstream to alternatively spliced exons (Lim and Sharp 1998; Deguillien et al. 2001).

Figure 3

Human–mouse alignment of the KCND3 gene, corresponding to RefSeq NM_004980 (from VISTA browser, http://pipeline.lbl.gov/vistabrowser/). x-axis: The nucleotide coordinates on human chromosome 1, according to the assembly version of the human genome from June 2002. y-axis: The level of conservation between the human genome and the corresponding mouse genome, according to the MGSCv3 assembly version of the mouse genome. (A) Blue bars above the conservation area correspond to annotated exons 4–8 of KCND3. Blue areas within the conservation graph mark exons; orange areas mark conserved nonexonic sequences. The exon marked with an arrow (exon 6) is an alternatively spliced one; the others are constitutively spliced exons. (B) Enlarged view of the conservation graphs of the alternatively spliced exon (exon 6), and one of the constitutively spliced exons (exon 4) is presented to show the relative lengths of the conserved areas near the exons. (C) Human, mouse, and rat alignment of exon 6, as well as the 100 bases upstream and downstream of the exon. Exon sequence is bold; asterisks mark identity in all three organisms. Bold and underline mark the hexamer TGCATG, which previously showed the ability to regulate alternative splicing when found in introns downstream to alternatively spliced exons (Lim and Sharp 1998; Deguillien et al. 2001).