RNA secondary structure in mutually exclusive splicing

Journal name:
Nature Structural & Molecular Biology
Volume:
18,
Pages:
159–168
Year published:
DOI:
doi:10.1038/nsmb.1959
Received
Accepted
Published online

Abstract

Mutually exclusive splicing is a regulated means to generate protein diversity, but the underlying mechanisms are poorly understood. Here comparative genome analysis revealed the built-in intronic elements for controlling mutually exclusive splicing of the 14-3-3ξ pre-mRNA. These elements are clade specific but are evolutionarily conserved at the secondary structure level. Combined evidence revealed the triple functions of these inter-intronic RNA pairings in synergistically ensuring the selection of only one of multiple exons, through activation of the proximal variable exon outside the loop by the approximation of cis elements, and simultaneous repression of the exon within the loop, in combination with the physical competition of RNA pairing. Additionally, under this model, we also deciphered a similar structural code in exon clusters 4 and 9 of Dscam (38,016 isoforms) and Mhc (480 isoforms). Our findings suggest a broadly applicable mechanism to ensure mutually exclusive splicing.

At a glance

Figures

  1. Phylogenetic arrangement of cis intronic elements in insect 14-3-3ξ genes.
    Figure 1: Phylogenetic arrangement of cis intronic elements in insect 14-3-3ξ genes.

    A schematic diagram of the partial pre-mRNA with constitutive exons depicted as black boxes, mutually exclusive exons as blue boxes, and introns as lines. Above are sequences of consensus intronic elements (IE) for different species taxa (see Supplementary Table 1 for abbreviations). The most identical nucleotides at each position are shown in different colors. Docking sites (marked by crowns) were reverse-complementary to an upstream selector sequence (marked by hearts).

  2. Mutually exclusive splicing is directed by competing RNA secondary structures.
    Figure 2: Mutually exclusive splicing is directed by competing RNA secondary structures.

    (a) Genomic organization of the 14-3-3ξ gene of D. melanogaster. Symbols used are the same as in Figure 1. Red, blue and yellow dotted lines represent splice products I, II and III, respectively. (b) The predicted RNA pairings of the 14-3-3ξ pre-mRNA. Mutations are introduced into dsRNA (M1–M4). Red half-circles and upside-down concave shapes represent compensatory double mutations (M13 and M34), and M2 (marked by blue square) is not reverse-complementary to M3. The arrow is depicted as activating the inclusion of the proximal exon. (c) Effects of competing RNA pairings were validated by disruptive mutations (M1–M4) and compensatory double mutations (M13 and M34). WT: wild-type; D1, D2, D3: IE1, IE2 or IEa deletion mutant. Data are expressed as mean ± s.d. from three independent experiments. (d) Effects of mutations on exon 5 selection. (e) Genomic organization and the RNA pairing of the 14-3-3ξ gene of A. mellifera, with the estimated equilibrium free energies (in kcal mol−1). (f) Analysis of the selection frequency of splicing variants in various tissues of flies and bees.

  3. RNA pairing controls the switching of mutually exclusive exons in B. mori 14-3-3ξ pre-mRNA.
    Figure 3: RNA pairing controls the switching of mutually exclusive exons in B. mori 14-3-3ξ pre-mRNA.

    (a) Genomic organization of the B. mori 14-3-3ξ gene. Symbols used are the same as in Figure 1. (b) Predicted RNA pairing of B. mori 14-3-3ξ pre-mRNA. (c) Effects of mutations on exon 5 inclusion are indicated for disruptive mutations (M1, M2) and compensatory double mutations (M12). (d) Effects of mutations on exon 5 selection. (e) Schematic diagrams of a series of mutant constructs. (f) Effect of RNA pairing on exon b inclusion. Data are expressed as mean ± s.d. from three independent experiments.

  4. Mutually exclusive splicing is regulated by base-pairing strength and inter-distance.
    Figure 4: Mutually exclusive splicing is regulated by base-pairing strength and inter-distance.

    (a) Predicted RNA pairing for the wild-type and a series of mutants (point mutations are shown in blue), with the estimated equilibrium free energies (in kcal mol−1). (b) The strength of RNA pairing modulated exon 5a selection by mutated analysis. Data are expressed as mean ± s.d. from three independent experiments. (c) Schematic diagrams of mutant minigene constructs with different looping distances. (d) Effects of looping distance on the choice of alternative exon.

  5. The effect of the distance between the 5′ splice site and the selectors on the efficiency of exon selection.
    Figure 5: The effect of the distance between the 5′ splice site and the selectors on the efficiency of exon selection.

    (a) Schematic diagrams of mutant minigene constructs. Nucleotide mutations, which destroyed the 5′ and 3′ splice sites of exon 5b, are indicated by arrows. A red cross in IE denotes the disrupting mutation; a green arrow depicts activating the inclusion of the proximal exon outside the loop; a green arrow plus a red cross marks complete abolishment of the inclusion; and a green arrow plus a red arrow marks the decrease in inclusion efficiency. (b) mRNA products from the minigenes (M1–M5) were analyzed by RT-PCR and variant-specific restriction digestion. (c) The effect of the distance between the 5′ splice site and the selectors on the efficiency of exon 5 inclusion and selection. Data are expressed as mean ± s.d. from three independent experiments. (d) Within-cluster intronic characteristics of the 14-3-3ξ genes were investigated in Drosophila, lepidopteran, coleopteran and hymenopteran species.

  6. RNA pairing interaction functions to approximate upstream and downstream sequences.
    Figure 6: RNA pairing interaction functions to approximate upstream and downstream sequences.

    (a) Schematic diagrams of minigene constructs used to test the importance of RNA secondary structure. Symbols used are the same as in Figure 1. (b) Effects of exon 5 selection by mimicking an RNA duplex. Data are expressed as mean ± s.d. from three independent experiments. (c) Overview of minigene constructs used to test the importance of approximating sequences. A cross in IE or IEa denotes the disrupting mutation. (d) Effects of exon 5 selection by a series of deletions and mutations.

  7. Similar structural codes within the exon clusters 4 and 9 of Dscam and Mhc gene.
    Figure 7: Similar structural codes within the exon clusters 4 and 9 of Dscam and Mhc gene.

    (a) Overview of the minigene constructs of Dscam. Constitutive exons (in black boxes), alternative exons (in colored boxes), docking sites and selectors and introns (lines) are shown. The most identical nucleotides with respect to docking sites and selector sequences are shown in different colors. (b) The predicted RNA pairings of Dscam pre-mRNA. (c) The inter-intronic pairings are conserved in Drosophila species. (d) Effects of RNA secondary structures were validated by disruptive mutations (M2, M3) and compensatory double mutations (M23). The band marked by “*” is a nonspecific RT-PCR product. (e,f) Effects of mutations on exon 4 inclusion (e) and selection (f) Data are expressed as mean ± s.d. from three independent experiments. (g) The arrangement of cis intronic elements in Drosophila Mhc. Symbols are the same as in Dscam. (h) RNA structural architecture was proposed to direct alternative splicing. The red circles, triangles and diamonds depict selector sequences for exon clusters 7, 9 and 11, respectively.

  8. Models for the control and regulation of mutually exclusive splicing.
    Figure 8: Models for the control and regulation of mutually exclusive splicing.

    (a) A model for the control of mutually exclusive splicing. In order for an exon 5 variant to be included in the 14-3-3ξ mRNA, the selector sequence (IE1, IE2) upstream of the exon may interact with the docking site (IEa) to form a splicing-activating complex. Conversely, exon 5c would be included if the docking site assumed a linear conformation without specific RNA pairing interactions, whereas exon 5a and exon 5b are not included. In no case are exon 5a, exon 5b and exon 5c included at the same time. The red and blue ovals depict the splicing repressors and activators, respectively. (b) A model for the regulation of mutually exclusive splicing. The choice of the alternative exon is regulated by overlapping base-pairing strength and inter-distance. The blue and red circles show the selector sequences and docking site, respectively.

Accession codes

Referenced accessions

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Author information

  1. These authors contributed equally to this work.

    • Yun Yang,
    • Leilei Zhan &
    • Wenjing Zhang

Affiliations

  1. Institute of Biochemistry, College of Life Sciences, Zhejiang University (Zijingang Campus), Hangzhou, Zhejiang, People's Republic of China.

    • Yun Yang,
    • Leilei Zhan,
    • Wenjing Zhang,
    • Feng Sun,
    • Wenfeng Wang,
    • Nan Tian,
    • Jingpei Bi,
    • Haitao Wang,
    • Dike Shi,
    • Yajian Jiang &
    • Yongfeng Jin
  2. Institute of Biochemistry, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, People's Republic of China.

    • Yaozhou Zhang

Contributions

Y.Y., W.Z., L.Z. and W.W. conducted mutational studies and splicing analyses; L.Z. collected, cloned and analyzed the nucleotide sequences; W.Z. was responsible for tissue-specific analyses of mutually exclusive splicing; F.S. conducted mutational studies and splicing analyses in silkworm; H.W., D.S. and Y. Jiang made the DNA constructs; N.T. and J.B. analyzed sequence data and RNA secondary structures. Y. Jin conceived of this project, designed the experiments, analyzed the data and wrote the manuscript; Y.Z. analyzed the data; all authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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