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A Segmental Genomic Duplication Generates a Functional Intron

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A Segmental Genomic Duplication Generates a Functional Intron

Uffe Hellsten et al. Nat Commun.

Abstract

An intron is an extended genomic feature whose function requires multiple constrained positions-donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers-that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half-a-billion years ago.

Figures

Figure 1
Figure 1. Proposed mechanism for intron birth.
Extant lancelet and lamprey ATP2A genes, and human and zebrafish ATP2A2 genes, are intronless in the region shown, reflecting the ancestral chordate condition, but human and zebrafish ATP2A1 genes are interrupted by an intron between the first and second nucleotides of codon G310 (coordinate with respect to human amino acid sequence of ATP2A1 isoform a). The peptide sequence is fully conserved, so only synonymous amino acid codon substitutions are seen in the nucleotide sequence. A segmental tandem duplication encompassing this region would produce a potential intron with consensus donor and acceptor splice sites, including a polypyrimidine tract. The sequence of the intronless human ATP2A2 gene is used in this example.
Figure 2
Figure 2. Reconstruction of duplicated splice sites undergoes splicing at low levels.
(a) Schematic diagram of mini-gene constructs transfected into HEK 293 and HeLa cells including ATP2A2 duplicated exon 8/9 (D), ATP2A2 Single (C1) and ATP2A2 Single with 6 bp insert (C2). The sequence corresponding to exon 8 is shaded light grey, exon 9 dark grey, 6 bp insert black and vector sequences are shown by dashed lines. (b) Diagram of the RNase protection probe along with annotations of what sequences each part of the probe will hybridize to. (c) Schematic representation of potential mRNA species from the transfections and the corresponding RNA probe fragments that their presence will lead to in the RNase protection assay. (d) Phosphorimage of RNase protection assay products from HEK 293 cells with DNA size marker sizes (in nt ssDNA) indicated on the right, and what size RNA fragments the protected probe bands correspond to on the left (ssRNA). Transfections were performed in triplicate. (e) Phosphorimage of RNase protection assay products from HeLa cells with DNA size marker sizes (in nt ssDNA) indicated on the right, and what size RNA fragments the protected probe bands correspond to on the right (ssRNA). Transfections were performed in triplicate.
Figure 3
Figure 3. Plasmids do not undergo DNA rearrangment during transfection.
(a) Schematic of DNA mini-gene constructs used in transfections as described in Figure 2a. Restriction sites are shown along with sizes of DNA digestion fragments. (b) Agarose gels showing diagnostic digests of DNA plasmids recovered from HEK 293 cells transfected with ATP2A2 duplicated 8/9 with insert (D). Control digests from untransfected plasmids are shown; ATP2A2 duplicated 8/9 with insert (D), ATP2A2 single (C1) and ATP2A2 single with 6 bp insert (C2), along with DNA size markers (m). All the recovered DNA plasmids were the same size as the transfected DNA plasmid ATP2A2 duplicated 8/9 with insert (D).

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