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Sequence features involved in the mechanism of 3' splice junction wobbling.

Tsai KW, Chan WC, Hsu CN, Lin WC - BMC Mol. Biol. (2010)

Bottom Line: By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site.Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices.Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. wenlin@ibms.sinica.edu.tw

ABSTRACT

Background: Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms.

Results: By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site. Although a lower frequency of alternative splicing occurs within the intronic region (alternative splicing at the proximal AG) than in the exonic region (alternative splicing at the distal AG), alternative AG sites located within the intronic region show stronger potential as the acceptor. These observations revealed that the choice of 3' splice sites during 3' splicing junction wobbling could depend on the distance between the duplicated AG and the branch point site (BPS). Further mutagenesis experiments demonstrated that the distance of AG-to-AG and BPS-to-AG can greatly influence 3' splice site selection. Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices.

Conclusion: Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function. In this study, we showed that occurrence of 3' wobble splicing occurs in a distance-dependent manner and that most of this wobble splicing is probably caused by steric hindrance from a factor bound at the neighboring tandem motif sequence.

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Distribution of 3' alternative splicing at positions ranging from two to twenty nucleotides from the dominant splice site. Schematic representation of three groups of 3' alternative splicing occurring close to the dominant site: 3'-AS_dominant site (black line), 3'-AS_proximal site (blue dashed line) and distal site (red dashed line). The brown squares indicate the total number of alternative splices at the 3' splice sites. Alternative splicing at the proximal splice site (intronic splice site) and the distal splice site (exonic splice site) is indicated by triangles and circles. The numbers of each of the three groups of alternative splicing occurring within six nucleotides of the dominant splice site are indicated in the top panel and the percentage in each group was determined as (number of alternative splices within six nucleotides of the dominant site)/(total number of alternative splices within 20 nucleotides of the dominant splice site) × 100. The red asterisks indicate that alternative splicing occurred at in-frame sites.
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Figure 1: Distribution of 3' alternative splicing at positions ranging from two to twenty nucleotides from the dominant splice site. Schematic representation of three groups of 3' alternative splicing occurring close to the dominant site: 3'-AS_dominant site (black line), 3'-AS_proximal site (blue dashed line) and distal site (red dashed line). The brown squares indicate the total number of alternative splices at the 3' splice sites. Alternative splicing at the proximal splice site (intronic splice site) and the distal splice site (exonic splice site) is indicated by triangles and circles. The numbers of each of the three groups of alternative splicing occurring within six nucleotides of the dominant splice site are indicated in the top panel and the percentage in each group was determined as (number of alternative splices within six nucleotides of the dominant site)/(total number of alternative splices within 20 nucleotides of the dominant splice site) × 100. The red asterisks indicate that alternative splicing occurred at in-frame sites.

Mentions: GTNGT- and NAGNAG-based wobble splicing events are widespread in the human genome, especially the 3' wobble splicing event that, according to an expressed sequence tag (EST) database survey, occurs in 30% of human genes and is active in at least 5% of genes [7]. In order to investigate further the distribution of wobble splicing events within the human genome, we have performed an in-depth computational analysis using a well known database--the Alternative Splicing Database (ASD) [20]. The ASD is derived from EST entries and reports the use of alternative splicing sites within the human transcriptome. In this study, we extracted 7,400 and 8,223 explicit alternative sites in 5' and 3' junctions, respectively, removing all ambiguous and conflicting instances. In the analysis of wobble splicing usage, the splice sites used for generating the highest numbers of transcripts according to the ASD dataset were defined as dominant splice sites. Additional putative alternate splice sites located around dominant splice sites are defined as proximal sites and distal sites as indicated in Figure 1 and 2. In each splicing instance, the number of ESTs occurring in relation to the distance from the dominant splice site was tabulated as shown in Figure 1 and 2. Our data indicated that short-distance wobble splicing events at the 3' tandem NAGNAG motif occur with a higher frequency than 5' GTNGT sites (1,999 cases vs. 782 cases), because the 3' end of an intron has a more intricate set of regulatory elements (Figure 1 and 2). Interestingly, 3' alternative splicing at a tandem acceptor has a higher frequency if the distance between the two tandem splice sites is less than six nucleotides. The frequency decreases significantly when the distance from the dominant splice site increases (> 6 nucleotides). However, alternative acceptor choice occurring within six nucleotides of the dominant splice site is more frequent in the exonic region (3'-AS_distal) than the intronic (3'-AS_proximal) region (3'-AS_distal: 1,605 cases; 3'-AS_proximal: 394 cases) (Figure 1). This is probably because the PPT preceding AG is a required feature for the splicing process; this therefore reduces the occurrence of 3'-AS_proximal site choices. Surprisingly, we have observed a higher ratio of minor isoforms in the 3'-AS_proximal AG selection dataset than in the 3'-AS_distal AG dataset even though the alternative AG is closer to the dominant AG (AG-to-AG distance < 10 nucleotides) (Figure 3). Although we observed a lower frequency of alternative splicing within the intronic region (3'-AS_proximal AG) than in the exonic region (3'-AS_distal AG), the alternative AG in the intronic region does possess greater potential as an acceptor site. Presumably, the spliceosome complex recognizes the branch point and scans downstream for the first AG by the hypothesized linear scanning mechanism [21-24]. The above results indicated that the process of 3' splice site selection may depend on the distance between the proximal AG and the distal AG (AG-to-AG), or the BPS and tandem splice sites (BPS-to-AG). Interestingly, we did not observe this feature involved in 5' short-distance wobble splicing. According to the ASD screening data, only one high frequency of 5' alternative splicing occurs at four nucleotides upstream or downstream from the dominant splice site [GT(N)2 GT: 403 cases] (Figure 2). Previous studies indicated that such bias could result from the strong U1 snRNP-binding conserved sequence at 5' splice sites [9,25]. We further analyzed the distribution of 5'-AS in UTR and CDS and found that 5'-GT(N)2GT alternative splicing frequently occurs in UTR region (~ 50%) (Additional file 1, Figure S1). While wobble splicing occurred in UTR region, it would not alter open reading frame and could escape from NMD degradation. Therefore, it is a possible explanation for overabundance of the 5'-GT(N)2GT wobble splicing.


Sequence features involved in the mechanism of 3' splice junction wobbling.

Tsai KW, Chan WC, Hsu CN, Lin WC - BMC Mol. Biol. (2010)

Distribution of 3' alternative splicing at positions ranging from two to twenty nucleotides from the dominant splice site. Schematic representation of three groups of 3' alternative splicing occurring close to the dominant site: 3'-AS_dominant site (black line), 3'-AS_proximal site (blue dashed line) and distal site (red dashed line). The brown squares indicate the total number of alternative splices at the 3' splice sites. Alternative splicing at the proximal splice site (intronic splice site) and the distal splice site (exonic splice site) is indicated by triangles and circles. The numbers of each of the three groups of alternative splicing occurring within six nucleotides of the dominant splice site are indicated in the top panel and the percentage in each group was determined as (number of alternative splices within six nucleotides of the dominant site)/(total number of alternative splices within 20 nucleotides of the dominant splice site) × 100. The red asterisks indicate that alternative splicing occurred at in-frame sites.
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Related In: Results  -  Collection

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Figure 1: Distribution of 3' alternative splicing at positions ranging from two to twenty nucleotides from the dominant splice site. Schematic representation of three groups of 3' alternative splicing occurring close to the dominant site: 3'-AS_dominant site (black line), 3'-AS_proximal site (blue dashed line) and distal site (red dashed line). The brown squares indicate the total number of alternative splices at the 3' splice sites. Alternative splicing at the proximal splice site (intronic splice site) and the distal splice site (exonic splice site) is indicated by triangles and circles. The numbers of each of the three groups of alternative splicing occurring within six nucleotides of the dominant splice site are indicated in the top panel and the percentage in each group was determined as (number of alternative splices within six nucleotides of the dominant site)/(total number of alternative splices within 20 nucleotides of the dominant splice site) × 100. The red asterisks indicate that alternative splicing occurred at in-frame sites.
Mentions: GTNGT- and NAGNAG-based wobble splicing events are widespread in the human genome, especially the 3' wobble splicing event that, according to an expressed sequence tag (EST) database survey, occurs in 30% of human genes and is active in at least 5% of genes [7]. In order to investigate further the distribution of wobble splicing events within the human genome, we have performed an in-depth computational analysis using a well known database--the Alternative Splicing Database (ASD) [20]. The ASD is derived from EST entries and reports the use of alternative splicing sites within the human transcriptome. In this study, we extracted 7,400 and 8,223 explicit alternative sites in 5' and 3' junctions, respectively, removing all ambiguous and conflicting instances. In the analysis of wobble splicing usage, the splice sites used for generating the highest numbers of transcripts according to the ASD dataset were defined as dominant splice sites. Additional putative alternate splice sites located around dominant splice sites are defined as proximal sites and distal sites as indicated in Figure 1 and 2. In each splicing instance, the number of ESTs occurring in relation to the distance from the dominant splice site was tabulated as shown in Figure 1 and 2. Our data indicated that short-distance wobble splicing events at the 3' tandem NAGNAG motif occur with a higher frequency than 5' GTNGT sites (1,999 cases vs. 782 cases), because the 3' end of an intron has a more intricate set of regulatory elements (Figure 1 and 2). Interestingly, 3' alternative splicing at a tandem acceptor has a higher frequency if the distance between the two tandem splice sites is less than six nucleotides. The frequency decreases significantly when the distance from the dominant splice site increases (> 6 nucleotides). However, alternative acceptor choice occurring within six nucleotides of the dominant splice site is more frequent in the exonic region (3'-AS_distal) than the intronic (3'-AS_proximal) region (3'-AS_distal: 1,605 cases; 3'-AS_proximal: 394 cases) (Figure 1). This is probably because the PPT preceding AG is a required feature for the splicing process; this therefore reduces the occurrence of 3'-AS_proximal site choices. Surprisingly, we have observed a higher ratio of minor isoforms in the 3'-AS_proximal AG selection dataset than in the 3'-AS_distal AG dataset even though the alternative AG is closer to the dominant AG (AG-to-AG distance < 10 nucleotides) (Figure 3). Although we observed a lower frequency of alternative splicing within the intronic region (3'-AS_proximal AG) than in the exonic region (3'-AS_distal AG), the alternative AG in the intronic region does possess greater potential as an acceptor site. Presumably, the spliceosome complex recognizes the branch point and scans downstream for the first AG by the hypothesized linear scanning mechanism [21-24]. The above results indicated that the process of 3' splice site selection may depend on the distance between the proximal AG and the distal AG (AG-to-AG), or the BPS and tandem splice sites (BPS-to-AG). Interestingly, we did not observe this feature involved in 5' short-distance wobble splicing. According to the ASD screening data, only one high frequency of 5' alternative splicing occurs at four nucleotides upstream or downstream from the dominant splice site [GT(N)2 GT: 403 cases] (Figure 2). Previous studies indicated that such bias could result from the strong U1 snRNP-binding conserved sequence at 5' splice sites [9,25]. We further analyzed the distribution of 5'-AS in UTR and CDS and found that 5'-GT(N)2GT alternative splicing frequently occurs in UTR region (~ 50%) (Additional file 1, Figure S1). While wobble splicing occurred in UTR region, it would not alter open reading frame and could escape from NMD degradation. Therefore, it is a possible explanation for overabundance of the 5'-GT(N)2GT wobble splicing.

Bottom Line: By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site.Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices.Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. wenlin@ibms.sinica.edu.tw

ABSTRACT

Background: Alternative splicing is an important mechanism mediating the diversified functions of genes in multicellular organisms, and such event occurs in around 40-60% of human genes. Recently, a new splice-junction wobbling mechanism was proposed that subtle modifications exist in mRNA maturation by alternatively choosing at 5'- GTNGT and 3'- NAGNAG, which created single amino acid insertion and deletion isoforms.

Results: By browsing the Alternative Splicing Database information, we observed that most 3' alternative splice site choices occur within six nucleotides of the dominant splice site and the incidence significantly decreases further away from the dominant acceptor site. Although a lower frequency of alternative splicing occurs within the intronic region (alternative splicing at the proximal AG) than in the exonic region (alternative splicing at the distal AG), alternative AG sites located within the intronic region show stronger potential as the acceptor. These observations revealed that the choice of 3' splice sites during 3' splicing junction wobbling could depend on the distance between the duplicated AG and the branch point site (BPS). Further mutagenesis experiments demonstrated that the distance of AG-to-AG and BPS-to-AG can greatly influence 3' splice site selection. Knocking down a known alternative splicing regulator, hSlu7, failed to affect wobble splicing choices.

Conclusion: Our results implied that nucleotide distance between proximal and distal AG sites has an important regulatory function. In this study, we showed that occurrence of 3' wobble splicing occurs in a distance-dependent manner and that most of this wobble splicing is probably caused by steric hindrance from a factor bound at the neighboring tandem motif sequence.

Show MeSH