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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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3' acceptor site selection depends on the AG-to-AG and BPS-to-AG distances. NM_014226-E6-I6-E7 (RAGE) (A) and NM_015179-E32-I32-E33 (RRP12) (B) minigene vectors containing four different distances between the proximal and distal AG, CAG(C)1AG, CAG(C)2AG; CAG(C)3AG and CAG(C)4AG (construction as shown in a panels). These minigenes were transfected into a HeLa cell line, and after 48 h total RNA and alternative splicing patterns were assayed as mentioned in the Materials and Methods. The expression profiles of these minigenes are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. (C) and (D) The BPS-to-AG distance of NM_015179-E32-I32-E33 was extended by inserting cytosines into the PPT region. These constructs are shown in the a panels and their expression profiles are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. The arrowheads and arrows indicate the use of distal and proximal AG, respectively. The percentage of wobble splicing isoforms is shown at the top of each b panel and the relative use of each AG is indicated by the thickness of the underlining in each a panel.
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Figure 5: 3' acceptor site selection depends on the AG-to-AG and BPS-to-AG distances. NM_014226-E6-I6-E7 (RAGE) (A) and NM_015179-E32-I32-E33 (RRP12) (B) minigene vectors containing four different distances between the proximal and distal AG, CAG(C)1AG, CAG(C)2AG; CAG(C)3AG and CAG(C)4AG (construction as shown in a panels). These minigenes were transfected into a HeLa cell line, and after 48 h total RNA and alternative splicing patterns were assayed as mentioned in the Materials and Methods. The expression profiles of these minigenes are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. (C) and (D) The BPS-to-AG distance of NM_015179-E32-I32-E33 was extended by inserting cytosines into the PPT region. These constructs are shown in the a panels and their expression profiles are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. The arrowheads and arrows indicate the use of distal and proximal AG, respectively. The percentage of wobble splicing isoforms is shown at the top of each b panel and the relative use of each AG is indicated by the thickness of the underlining in each a panel.

Mentions: Next, we determined whether the physical AG-to-AG and BPS-to-AG distance was involved in splice site selection. We constructed two minigene constructs both containing strong 3' wobble splicing tandem motifs (CAGCAG). According to EST information, one gene (RAGE: NN_014226) preferentially uses the proximal AG site (proximal/distal: 61.9%/38.1%) and another gene (RRP12: NM_015179) preferentially uses the distal AG site (proximal/distal: 7.6%/92.4%). As shown in Figure 5A and 3B, we inserted cytosine residues to increase the distance between proximal AG and distal AG. This makes the proximal AG become more competitive than the distal AG sites. Gradually increasing the number of cytosine residues to four in two minigenes caused the proximal AG to be used almost exclusively. Such results support the previous observation that nucleotide distance between proximal and distal AG affects the wobble splicing choice (Figure 5A and 5B). A short distance of six nucleotides or less could create competition between distal and proximal AG sites (Figure 5A and 5B). Next, we further examined the AG site choice of the NM_015179 minigene by increasing the BPS-to-AG distance. These minigene constructs with varied BPS-to-AG distances were introduced into HeLa cells and splicing patterns were analyzed by a capillary electrophoresis approach. As shown in Figure 5B, C and 5D, increasing the BPS-to-AG nucleotide number could significantly reinforce the selection of the proximal AG splice site. When the distance between tandem AG splice sites is reduced to less than five nucleotides, the proximal AG cannot completely compete with the distal AG because of the increased BPS-to-AG distance (Figure 5B, C and 5D). This implies that the BPS-to-AG distance can also affect splice site selection, but also that it cannot influence the occurrence of wobble splicing at close tandem motifs. In conclusion, the choice of tandem acceptor sites during wobble splicing is under the influence of both AG-to-AG and BPS-to-AG distance.


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

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

3' acceptor site selection depends on the AG-to-AG and BPS-to-AG distances. NM_014226-E6-I6-E7 (RAGE) (A) and NM_015179-E32-I32-E33 (RRP12) (B) minigene vectors containing four different distances between the proximal and distal AG, CAG(C)1AG, CAG(C)2AG; CAG(C)3AG and CAG(C)4AG (construction as shown in a panels). These minigenes were transfected into a HeLa cell line, and after 48 h total RNA and alternative splicing patterns were assayed as mentioned in the Materials and Methods. The expression profiles of these minigenes are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. (C) and (D) The BPS-to-AG distance of NM_015179-E32-I32-E33 was extended by inserting cytosines into the PPT region. These constructs are shown in the a panels and their expression profiles are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. The arrowheads and arrows indicate the use of distal and proximal AG, respectively. The percentage of wobble splicing isoforms is shown at the top of each b panel and the relative use of each AG is indicated by the thickness of the underlining in each a panel.
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Related In: Results  -  Collection

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Figure 5: 3' acceptor site selection depends on the AG-to-AG and BPS-to-AG distances. NM_014226-E6-I6-E7 (RAGE) (A) and NM_015179-E32-I32-E33 (RRP12) (B) minigene vectors containing four different distances between the proximal and distal AG, CAG(C)1AG, CAG(C)2AG; CAG(C)3AG and CAG(C)4AG (construction as shown in a panels). These minigenes were transfected into a HeLa cell line, and after 48 h total RNA and alternative splicing patterns were assayed as mentioned in the Materials and Methods. The expression profiles of these minigenes are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. (C) and (D) The BPS-to-AG distance of NM_015179-E32-I32-E33 was extended by inserting cytosines into the PPT region. These constructs are shown in the a panels and their expression profiles are indicated in the b panels and the relative percentage of the two isoforms was showed in the c panels. The arrowheads and arrows indicate the use of distal and proximal AG, respectively. The percentage of wobble splicing isoforms is shown at the top of each b panel and the relative use of each AG is indicated by the thickness of the underlining in each a panel.
Mentions: Next, we determined whether the physical AG-to-AG and BPS-to-AG distance was involved in splice site selection. We constructed two minigene constructs both containing strong 3' wobble splicing tandem motifs (CAGCAG). According to EST information, one gene (RAGE: NN_014226) preferentially uses the proximal AG site (proximal/distal: 61.9%/38.1%) and another gene (RRP12: NM_015179) preferentially uses the distal AG site (proximal/distal: 7.6%/92.4%). As shown in Figure 5A and 3B, we inserted cytosine residues to increase the distance between proximal AG and distal AG. This makes the proximal AG become more competitive than the distal AG sites. Gradually increasing the number of cytosine residues to four in two minigenes caused the proximal AG to be used almost exclusively. Such results support the previous observation that nucleotide distance between proximal and distal AG affects the wobble splicing choice (Figure 5A and 5B). A short distance of six nucleotides or less could create competition between distal and proximal AG sites (Figure 5A and 5B). Next, we further examined the AG site choice of the NM_015179 minigene by increasing the BPS-to-AG distance. These minigene constructs with varied BPS-to-AG distances were introduced into HeLa cells and splicing patterns were analyzed by a capillary electrophoresis approach. As shown in Figure 5B, C and 5D, increasing the BPS-to-AG nucleotide number could significantly reinforce the selection of the proximal AG splice site. When the distance between tandem AG splice sites is reduced to less than five nucleotides, the proximal AG cannot completely compete with the distal AG because of the increased BPS-to-AG distance (Figure 5B, C and 5D). This implies that the BPS-to-AG distance can also affect splice site selection, but also that it cannot influence the occurrence of wobble splicing at close tandem motifs. In conclusion, the choice of tandem acceptor sites during wobble splicing is under the influence of both AG-to-AG and BPS-to-AG distance.

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