A mechanism for a single nucleotide intron shift

Abstract

Spliceosomal introns can occupy nearby rather than identical positions in orthologous genes (intron sliding or shifting). Stwintrons are complex intervening sequences, where an 'internal' intron interrupts one of the sequences essential for splicing, generating after its excision, a newly formed canonical intron defined as 'external'. In one experimentally demonstrated configuration, two alternatively excised internal introns, overlapping by one G, disrupt respectively the donor and the acceptor sequence of an external intron, leading to mRNAs encoding identical proteins. In a gene encoding a DHA1 antiporter in Pezizomycotina, we find a variety of predicted intron configurations interrupting the DNA stretch encoding a conserved peptidic sequence. Some sport a stwintron where the internal intron interrupts the donor of the external intron (experimentally confirmed for Aspergillus nidulans). In others, we found and demonstrate (for Trichoderma reesei) alternative, overlapping internal introns. Discordant canonical introns, one nt apart, are present in yet other species, exactly as predicted by the alternative loss of either of the internal introns at the DNA level from an alternatively spliced stwintron. An evolutionary pathway of 1 nt intron shift, involving an alternatively spliced stwintron intermediate is proposed on the basis of the experimental and genomic data presented.

Figure 1.

A mechanism for a 1-nt intron shift involving the ancestral existence of an alternatively spliced [D1,2]/[A2,3] stwintron intermediate. (A) Structure and nomenclature of stwintrons relevant to this study. Intronic nt are in lower case letter: nt of the internal intron (light gray) in red, and nt of the external intron (dark gray) in blue lettering. The conserved terminal sequences essential for spliceosomal intron excision are indicated: D, donor sequence (gt-) at the 5′ splice site, and A, acceptor sequence (-ag) at the 3′ splice site. In a [D1,2] stwintron, an internal U2 intron interrupts the donor element of an external U2 intron between the first and the second nt. In a [A2,3] stwintron, the internal intron is nested in the (3-nt) acceptor element of the external intron between the second and the third nt. The G upstream the most 5′ donor is a characteristic feature of the [D1,2] stwintron but is exonic for the [A2,3] stwintron; The G downstream the most 3′ acceptor is a characteristic feature of the [A2,3] stwintron but is exonic for the [D1,2] stwintron. (B) A mechanism for the evolution of neighboring discordant intron positions. An alternatively spliced [D1,2]/[A2,3] stwintron occurs. When subsequently its [A2,3] internal intron is lost, the remaining canonical intron is localized at the 3′ of neighboring, alternative exon fusion sites utilized by the ancestor [D1,2]/[A2,3] stwintron. However, if its [D1,2] internal intron is lost, the de novo canonical intron is localized at the 5′ of those exon fusion sites. The two U2 introns resulting from mutually exclusive internal intron loss events at the DNA level are shown respectively at the top and the bottom, the two intron positions being called 5′ and 3′.

Figure 2.

Phylogenetic relations amongst the 89 Pezizomycotina DHA1 proteins most closely related to Aspergillus nidulans DhaoS. The figure shows a well-defined clade extracted from much larger Maximum Likelihood tree of Pezizomycotina DHA1 proteins that are at least 45% identical to DhaoS (see ‘Results’ section). Approximate Likelihood Ratio Test values are given at each node. Proteins encoded by 36 orthologous genes that contain a strict [D1,2] stwintron are tagged _D and shown in black lettering. Proteins encoded by 28 orthologous genes that harbor an alternatively spliced, [D1,2]/[A2,3] stwintron at the same position are tagged _A and shown in blue lettering. The proteins encoded by nine genes that harbor a canonical phase-2 intron are tagged _C5 and printed in red lettering while those encoded by the two orthologous genes that carry a phase-0 intron are tagged _C3 and printed in green lettering. The 16 DHA1 proteins in the outgroup (labeled _P and shown in gray lettering) are encoded by intron-less ‘nearest paralog’ genes; in Oidiodendron maius, this gene is duplicated (_Pa and _Pb, respectively). The 21 proteins in bold lettering are encoded by orthologous genes that comprise an additional standard intron, 329 nt downstream of the [D1,2] stwintron position.

Figure 3.

Relevant sequences of the DHA1 genes orthologous to Aspergillus nidulans dhaoS that harbor discordant canonical introns rather than a stwintron. The discordant introns interrupt the DNA encoding the conserved amino acid sequence WGPLSELY. Coding nt are in capitals. Species abbreviations: As.s, Aspergillus sclerotiorum; As.z, Aspergillus zonatus; Pe.e, Penicillium expansum; Pe.j, Penicillium janthinellum; Pe.r, Penicillium raistrickii; Pe.c, Penicillium citrinum; Ra.q, Raffaelea quercivora; Ch.l, Chalara longipes; Co.l, Coniochaeta ligniaria. The corresponding sequence in Penicillium aurantiogriseum NRRL 62431 (accession ALJY00000000) is identical to that in P. expansum T01 (AYHP00000000). The corresponding sequence in Hymenoscyphus varicosporoides CBS 651.66 (LLCF00000000) is identical to that in P. citrinum DSM 1997 (LKUP00000000). The 1-nt intron shift is indicated by highlighting in green the third nt of the codon for the first Leu in WGPLSELY. The conserved sequence element around the putative lariat branch point A (6 nt) is highlighted in yellow.