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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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The effect of hSlu7 on tandem splice site selection. HeLa cells were transfected with the RNAi oligonucleotide directed against either hSlu7 or luciferase (control). (A) After 48 h, total protein was extracted, separated by 10% SDS-PAGE, transferred to a membrane and probed for hSlu7 and actin. (B) The total RNA was collected, followed by RT-PCR using specific primers. The alternative splicing of the DDO-1 gene was affected by the hSlu7 protein concentration (positive control). GAPDH, a housekeeping gene, was amplified in each sample to confirm that approximately the same amount of cDNA was used for each reaction. (C) and (D) Analysis of 3' wobble splicing of three endogenous genes (FOXM1: NM_021953, PGAM5: NM_138575 and METTL9: NM_016025) and four minigenes (RAGE: NM_014226, SIPA1L1: NM_015556, ARID1A: NM_018450 and AG(T)9CAG) in hSlu7 knockdown HeLa cells using capillary electrophoresis (upper panel). The relative percentage of the two isoforms was calculated using GeneScan 3.7 (lower panel).
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Figure 4: The effect of hSlu7 on tandem splice site selection. HeLa cells were transfected with the RNAi oligonucleotide directed against either hSlu7 or luciferase (control). (A) After 48 h, total protein was extracted, separated by 10% SDS-PAGE, transferred to a membrane and probed for hSlu7 and actin. (B) The total RNA was collected, followed by RT-PCR using specific primers. The alternative splicing of the DDO-1 gene was affected by the hSlu7 protein concentration (positive control). GAPDH, a housekeeping gene, was amplified in each sample to confirm that approximately the same amount of cDNA was used for each reaction. (C) and (D) Analysis of 3' wobble splicing of three endogenous genes (FOXM1: NM_021953, PGAM5: NM_138575 and METTL9: NM_016025) and four minigenes (RAGE: NM_014226, SIPA1L1: NM_015556, ARID1A: NM_018450 and AG(T)9CAG) in hSlu7 knockdown HeLa cells using capillary electrophoresis (upper panel). The relative percentage of the two isoforms was calculated using GeneScan 3.7 (lower panel).

Mentions: The cis elements and trans-acting factors are involved in precise recognition of the splice sites during the splicing process [26]. Therefore, we first investigated whether trans-acting splicing factor was involved in 3' wobble splicing. The splicing factor Slu7 has been shown to affect 3' AG selection during step II of the splicing process in vitro and has been suggested to affect alternative splicing choice in vivo [19,24,27]. To determine whether the trans protein factor hSlu7 is involved in 3' splice site selection in wobble splicing events, we used an RNAi approach to knockdown hSlu7. hSlu7 RNAi treatment led to a 90% reduction in hSlu7 protein level and affected the exon inclusion/skipping ratio of a target gene, D-aspartate oxidase (DDO), changing it from mostly exon skipping to inclusion. This result indicated that as expected, the RNAi-mediated reduction in the nuclear level of hSlu7 had functional consequences in alternative splice choice (Figure 4A and 4B). However, the patterns of 3'-NAGNAG-based wobble splicing were not significantly altered by hSlu7 knockdown (Figure 4C), which suggests that hSlu7 is dispensable for 3'-NAGNAG-based alternative splicing in very closely linked tandem motifs (within three nucleotides). This is observed in endogenous genes as well as in transfected minigene constructs. Previous studies showed that aberrant AG site selection could be demonstrated by in vitro splicing assay using ΔhSlu7 extracts, while the duplicated AG is located upstream or downstream of the normal AG (AG-to-AG distance: from 6 to 12 nucleotides) [27]. To further determine whether the distance between AG-to-AG is a critical factor in slu7 deciding 3' splice-site choice and the efficiency of our hSlu7 RNAi treatment, we examined the same AG(N)9CAG construct used from previous publication (11AG/23AG construct) [27]. As shown in Figure 4D, the use of the distal AG site (23AG) was decreased and proximal AG site (11AG) was significantly activated by reducing hSlu7 protein level in si-hSlu7 transfected cells. This result confirmed that the AG site choice modulation effects of hSlu7 and appropriated distance between duplicate AGs might be needed for 3' splice sites selection by the Slu7 protein. From our bioinformatic and experimental results, the very short distance (three nucleotides) between proximal and distal AG sites might not be modulated by Slu7 alone.


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

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

The effect of hSlu7 on tandem splice site selection. HeLa cells were transfected with the RNAi oligonucleotide directed against either hSlu7 or luciferase (control). (A) After 48 h, total protein was extracted, separated by 10% SDS-PAGE, transferred to a membrane and probed for hSlu7 and actin. (B) The total RNA was collected, followed by RT-PCR using specific primers. The alternative splicing of the DDO-1 gene was affected by the hSlu7 protein concentration (positive control). GAPDH, a housekeeping gene, was amplified in each sample to confirm that approximately the same amount of cDNA was used for each reaction. (C) and (D) Analysis of 3' wobble splicing of three endogenous genes (FOXM1: NM_021953, PGAM5: NM_138575 and METTL9: NM_016025) and four minigenes (RAGE: NM_014226, SIPA1L1: NM_015556, ARID1A: NM_018450 and AG(T)9CAG) in hSlu7 knockdown HeLa cells using capillary electrophoresis (upper panel). The relative percentage of the two isoforms was calculated using GeneScan 3.7 (lower panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: The effect of hSlu7 on tandem splice site selection. HeLa cells were transfected with the RNAi oligonucleotide directed against either hSlu7 or luciferase (control). (A) After 48 h, total protein was extracted, separated by 10% SDS-PAGE, transferred to a membrane and probed for hSlu7 and actin. (B) The total RNA was collected, followed by RT-PCR using specific primers. The alternative splicing of the DDO-1 gene was affected by the hSlu7 protein concentration (positive control). GAPDH, a housekeeping gene, was amplified in each sample to confirm that approximately the same amount of cDNA was used for each reaction. (C) and (D) Analysis of 3' wobble splicing of three endogenous genes (FOXM1: NM_021953, PGAM5: NM_138575 and METTL9: NM_016025) and four minigenes (RAGE: NM_014226, SIPA1L1: NM_015556, ARID1A: NM_018450 and AG(T)9CAG) in hSlu7 knockdown HeLa cells using capillary electrophoresis (upper panel). The relative percentage of the two isoforms was calculated using GeneScan 3.7 (lower panel).
Mentions: The cis elements and trans-acting factors are involved in precise recognition of the splice sites during the splicing process [26]. Therefore, we first investigated whether trans-acting splicing factor was involved in 3' wobble splicing. The splicing factor Slu7 has been shown to affect 3' AG selection during step II of the splicing process in vitro and has been suggested to affect alternative splicing choice in vivo [19,24,27]. To determine whether the trans protein factor hSlu7 is involved in 3' splice site selection in wobble splicing events, we used an RNAi approach to knockdown hSlu7. hSlu7 RNAi treatment led to a 90% reduction in hSlu7 protein level and affected the exon inclusion/skipping ratio of a target gene, D-aspartate oxidase (DDO), changing it from mostly exon skipping to inclusion. This result indicated that as expected, the RNAi-mediated reduction in the nuclear level of hSlu7 had functional consequences in alternative splice choice (Figure 4A and 4B). However, the patterns of 3'-NAGNAG-based wobble splicing were not significantly altered by hSlu7 knockdown (Figure 4C), which suggests that hSlu7 is dispensable for 3'-NAGNAG-based alternative splicing in very closely linked tandem motifs (within three nucleotides). This is observed in endogenous genes as well as in transfected minigene constructs. Previous studies showed that aberrant AG site selection could be demonstrated by in vitro splicing assay using ΔhSlu7 extracts, while the duplicated AG is located upstream or downstream of the normal AG (AG-to-AG distance: from 6 to 12 nucleotides) [27]. To further determine whether the distance between AG-to-AG is a critical factor in slu7 deciding 3' splice-site choice and the efficiency of our hSlu7 RNAi treatment, we examined the same AG(N)9CAG construct used from previous publication (11AG/23AG construct) [27]. As shown in Figure 4D, the use of the distal AG site (23AG) was decreased and proximal AG site (11AG) was significantly activated by reducing hSlu7 protein level in si-hSlu7 transfected cells. This result confirmed that the AG site choice modulation effects of hSlu7 and appropriated distance between duplicate AGs might be needed for 3' splice sites selection by the Slu7 protein. From our bioinformatic and experimental results, the very short distance (three nucleotides) between proximal and distal AG sites might not be modulated by Slu7 alone.

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