An intronic signal for alternative splicing in the human genome

Abstract

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3' splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.

Figure 1. A diagram depicting In100 and its role in regulating casp-2.

As demonstrated by our previous studies , , sequential deletion and biochemical experiments reveal that In100 in casp-2 gene represses exon 9 inclusion by engaging non-productive interaction with the 5′ splice site of exon 9. Interactions between U1 snRNP associated with exon 9 at the 5′ splice site and complexes associated with In100, including U2 snRNP and PTB, block rather than promote the selection of this 5′ splice site. The In100 element is diagramed as the hatched box. Exons 8, 9 and 10 are depicted as E8, E9 and E10 in gray boxes. U1 snRNP and U2 snRNPs are illustrated as U1 and U2.

Figure 2. The evolutionary conservation of casp-2 In100 element and nucleotide composition of In100-like elements.

(A) Alignment between human and mouse casp-2 In100 elements shows 74% sequence identity. Putative branch site A is in bold and underlined (BS). The polypyrimidine tract in the decoy 3′splice site (Py)n and CAG are indicated. (B) The nucleotide composition is displayed based on the identified 4342 In100 elements.

Figure 3. The distribution of gene ontology of In100-containing genes.

The distribution of gene ontology terms of the In100-containing genes in (A) Biology Process, (B) Molecular Function and (C) Cell Component.

Figure 4. A biased distribution of transcripts favoring the downstream exons as revealed by EST analyses.

ESTs frequency of upstream (5′) and downstream (3′) exons. The sample sizes for analyzed In100-element flanking exons and control exon pairs are: 2728 and 23624, respectively.

Figure 5. The alternative splicing of In100-containing genes. (A) A diagram depicting the alternative splicing of In100-containing genes.

In100-containing genes were identified with three flanking exons (E1 to E3) from EST database, illustrated as E1-E2-In100-E3. 482 genes from EST database and an additional 44 genes from AltSplice Database, with a total of 526 different genes show exon 2 skipping. (B) A higher frequency of skipping of exon2 in In100-containing exon triplets revealed by EST analyses. Frequency of skipping of exon2 in In100-containing and In100-lacking exon triplets by EST evidence is: 36% and 26%, respectively. The sample sizes for analyzed In100-containing and control exon triplets are: 1470 and 15943, respectively.

Figure 6. Alternative splicing of caspase genes.

(A) A schematic illustration of In100-like elements in caspase genes and their alternative splicing patterns. Exons are depicted in rectangles, and In100 elements are illustrated as small gray boxes. The positions of regions encoding “QACRG” pentapeptide sequence are marked with “*”. (B) Alternative splicing of Caspase 2, 3, 8, 9 genes as detected by RT-PCR. Specific primers corresponding to the upstream and downstream exons were used in RT-PCR as described in Materials and Methods. The sizes of each exons are not drawn to scale. The positions of PCR primers are marked by arrows.