Alternative splicing regulation at tandem 3' splice sites

Abstract

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, approximately 25% are related to alternative choices of 3' and 5' splice sites. Surprisingly, half of all these events involve 3' splice sites that are exactly 3 nt apart. These tandem 3' splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of approximately 30 bp at the intronic regions flanking the 3' splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.

Figure 1

Distribution of indel (insertion/deletion) length in alternative 3′ (black, diamonds) and 5′ (gray, squares) splice sites revealing the high occurrence of exact 3 nt indels at alternative 3′ splice sites.

Figure 2

(A) Schematic diagram of the three groups of NAGNAG splice sites: NAGNAG 3′AS, proximal and distal. (B) Observed nucleotide frequency for the N of the proximal NAG site and (C) the N of the distal NAG site in the three different groups: NAGNAG 3′AS (white), ‘Proximal’ (black) and ‘Distal’ (stripedlines).

Figure 3

Human/mouse evolutionary conservation of NAGNAG acceptor sites. Percent of conservation was calculated for human/mouse pairwise alignments (hg17/mm5) derived from the UCSC site in three different datasets. (A) EST-confirmed NAGNAG 3′AS sites, (B) ‘Proximal’ group and (C) ‘Distal’ group. The numbers 1–6 indicate the nucleotide position at the NAGNAG splice site. The letters below each position indicate the base found in human. The heights of the letters represent the relative frequency of each nucleotide in a given position. The values below indicate the overall percent nucleotide conservation at that position. For positions (2, 5 and 3, 6), which are occupied in human by a single type of nucleotide, the percent conservation represents the conservation of the nucleotides (A or G) shown above. Graphic representations were carried out with the program Logos .

Figure 4

Human/mouse evolutionary conservation at the intronic flanking regions of exons with a NAGNAG sequence at the acceptor site. (A) Average conservation scores for 8 nt overlapping windows spanning 30 nt upstream the splicing acceptor and downstream the splicing donor at NAGNAG 3′AS sites (green), Skipped Exons (turquoise) and Constitutive spliced exons (black). (B) Conservation scores for conserved ‘Proximal’ and ‘Distal’ groups (plain, blue and red, respectively) and non-conserved ‘Proximal’ and ‘Distal’ groups (dashed, blue and red, respectively). Numbers in the X-axis represent the position of the first nucleotide of the window relative to the splice site. Values for each point i represent the average conservation of positions i − (i + 7).