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Cited by 108RNA Research and SplicingRNA and protein synthesis mechanismsRNA regulation and disease

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GC content around splice sites affects splicing through pre-mRNA secondary structures

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Zhang, Jing, Kuo, CC Jay, Chen, Liang·2011·BMC Genomics
DOI 10.1186/1471-2164-12-90PMC PMC3041747
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Abstract

BACKGROUND: Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens), mice (Mus musculus), fruit flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans) to further investigate this phenomenon. RESULTS: We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. CONCLUSION: All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

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Figure 1 Comparison of stability distribution of alternative splice sites and constitutive or skipped splice sites in humans. A and B are for the 5'ss comparison. C and D are for the 3' ss comparison. "alt" means alternative splice sites, "cons" means constitutive splice sites, and "skip" means skipped splice sites. Alternative splices sites tended to have more stable structures.
Figure 1 Comparison of stability distribution of alternative splice sites and constitutive or skipped splice sites in humans. A and B are for the 5'ss comparison. C and D are for the 3' ss comparison. "alt" means alternative splice sites, "cons" means constitutive splice sites, and "skip" means skipped splice sites. Alternative splices sites tended to have more stable structures.CC BY · PubMed Central
Figure 2 Distributions of the GC content of different splice sites in humans. Compared with constitutive and skipped splice sites, alternative splice sites were significantly enriched with GC (the p-values based on the Wilcoxon tests were all < 2.2 × 10-16).
Figure 2 Distributions of the GC content of different splice sites in humans. Compared with constitutive and skipped splice sites, alternative splice sites were significantly enriched with GC (the p-values based on the Wilcoxon tests were all < 2.2 × 10-16).CC BY · PubMed Central
Figure 3 GC content negatively correlates with energy in humans. A-C are for alternative, constitutive, and skipped 5'ss. D-F are for alternative, constitutive, and skipped 3'ss. The GC content in all splice sites in humans was negatively correlated with the predicted structural energy. The fitted regression lines and the R2 values are listed to show the goodness of fit.
Figure 3 GC content negatively correlates with energy in humans. A-C are for alternative, constitutive, and skipped 5'ss. D-F are for alternative, constitutive, and skipped 3'ss. The GC content in all splice sites in humans was negatively correlated with the predicted structural energy. The fitted regression lines and the R2 values are listed to show the goodness of fit.CC BY · PubMed Central
Figure 4 GC content difference explains structural stability difference. Boxplots of the energy are plotted (dots represent outliers). Alternative, constitutive, and skipped splice sites in humans had similar stability given the similar GC number (Wilcoxon tests for splice site energy comparison within each GC group, P > 0.05).
Figure 4 GC content difference explains structural stability difference. Boxplots of the energy are plotted (dots represent outliers). Alternative, constitutive, and skipped splice sites in humans had similar stability given the similar GC number (Wilcoxon tests for splice site energy comparison within each GC group, P > 0.05).CC BY · PubMed Central
Figure 5 Free energy and GC content comparison between tissue-specific and non-tissue-specific alternative donor and acceptor sites in humans with boxplots. A is for the energy comparison between tissue-specific and non-tissue-specific splice sites in humans. The average free energy at the donor sites was -46.25 kcals/mol for tissue-specific and -41.20 kcals/mol for non-tissue-specific donor sites (Wilcoxon test, P < 2.2 × 10-16). At the acceptor site, the average energy was -40.65 vs. -39.98 kcals/mol (Wilcoxon test, P = 0.04). B is for the GC content comparison. The average GC content for the tissue specific donor sites was 0.56, and decreased to 0.51 for non-tissue-specific ones (Wilcoxon test, P < 2.2 × 10-16). Similar results were obtained at the acceptor sites (0.53 vs 0.51, Wilcoxon test, P < 4.9 × 10-5).
Figure 5 Free energy and GC content comparison between tissue-specific and non-tissue-specific alternative donor and acceptor sites in humans with boxplots. A is for the energy comparison between tissue-specific and non-tissue-specific splice sites in humans. The average free energy at the donor sites was -46.25 kcals/mol for tissue-specific and -41.20 kcals/mol for non-tissue-specific donor sites (Wilcoxon test, P < 2.2 × 10-16). At the acceptor site, the average energy was -40.65 vs. -39.98 kcals/mol (Wilcoxon test, P = 0.04). B is for the GC content comparison. The average GC content for the tissue specific donor sites was 0.56, and decreased to 0.51 for non-tissue-specific ones (Wilcoxon test, P < 2.2 × 10-16). Similar results were obtained at the acceptor sites (0.53 vs 0.51, Wilcoxon test, P < 4.9 × 10-5).CC BY · PubMed Central
Figure 6 Comparison of GC percentage around real (red lines) and decoy (black) splice sites in humans. In general, the sequences around the real splice sites in humans were more GC enriched compared to the decoy ones (0.50 vs. 0.48 at the donor sites, 0.47 vs. 0.46 at the acceptor sites, Wilcoxon tests, P < 2.2 × 10-16). In addition, the exonic regions near the real splice sites were more enriched in GC compared to regions far away from the junctions, while no such enrichment has been observed near the decoy splice sites.
Figure 6 Comparison of GC percentage around real (red lines) and decoy (black) splice sites in humans. In general, the sequences around the real splice sites in humans were more GC enriched compared to the decoy ones (0.50 vs. 0.48 at the donor sites, 0.47 vs. 0.46 at the acceptor sites, Wilcoxon tests, P < 2.2 × 10-16). In addition, the exonic regions near the real splice sites were more enriched in GC compared to regions far away from the junctions, while no such enrichment has been observed near the decoy splice sites.CC BY · PubMed Central
Figure 7 Stability of native and permuted sequences in humans with boxplots. A and B are for alternative splice sites. C and D are for constitutive splice sites. E and F are for skipped splice sites. The permuted sequences ("_p1",white) that kept the first order nucleotide frequencies always had less stable structures than the native sequences ("_orig", black). The difference was significant but relatively small compared with the difference between different GC-number groups. However, when the dinucleotide frequencies were fixed ("_p2", grey), the permuted sequences exhibited comparable structural stability, indicating insignificant contribution from the nucleotide order.
Figure 7 Stability of native and permuted sequences in humans with boxplots. A and B are for alternative splice sites. C and D are for constitutive splice sites. E and F are for skipped splice sites. The permuted sequences ("_p1",white) that kept the first order nucleotide frequencies always had less stable structures than the native sequences ("_orig", black). The difference was significant but relatively small compared with the difference between different GC-number groups. However, when the dinucleotide frequencies were fixed ("_p2", grey), the permuted sequences exhibited comparable structural stability, indicating insignificant contribution from the nucleotide order.CC BY · PubMed Central

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Metadata

Authors
Zhang, J., Kuo, C. J., & Chen, L.
Affiliations
University of Southern California (US)
Corresponding author
Chen, Liang
ORCID
Venue
BMC Genomics
Publisher
Springer Science and Business Media LLC
Published
2011-01-31
Volume
12
Issue
1
Pages
90-90
ISSN
1471-2164 (ISSN-L)
DOI
10.1186/1471-2164-12-90
PMC
PMC3041747
OpenAlex
W2074860825
Cited by
108
References
34
Citations by year
2026: 11 · 2025: 10 · 2024: 8 · 2023: 8 · 2022: 6 · 2021: 7 · 2020: 3 · 2019: 12 · 2018: 4 · 2017: 5 · 2016: 3 · 2015: 7 · 2014: 9 · 2013: 6 · 2012: 7
FWCI
3.11
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Genomics (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • National Institutes of Health
  • National Human Genome Research Institute (P50HG002790)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
GC content, splice sites, alternative splicing, RNA secondary structure, pre-mRNA splicing, exon-intron junctions, tissue-specific splicing
MeSH terms
Animals, Base Composition, Drosophila, Humans, Nucleic Acid Conformation, RNA Precursors, RNA Splicing, Caenorhabditis elegans, Mice

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