Alternatively spliced, spliceosomal twin introns in Helminthosporium solani

• First demonstration of spliceosomal twin introns in Pleosporales (Dothideomycetes).
• Demonstration of internal intron interrupting acceptor domain of external intron.
• Demonstration of alternative splicing between overlapping internal introns.
• Demonstration of alternative splicing between stwintron and conventional intron.
• No obvious preference for alternative splicing pathways.

Spliceosomal twin introns, “stwintrons”, have been defined as complex intervening sequences that carry a second intron (“internal intron”) interrupting one of the conserved sequence domains necessary for their correct splicing via consecutive excision events. Previously, we have described and experimentally verified stwintrons in species of Sordariomycetes, where an “internal intron” interrupted the donor sequence of an “external intron”. Here we describe and experimentally verify two novel stwintrons of the potato pathogen Helminthosporium solani. One instance involves alternative splicing of an internal intron interrupting the donor domain of an external intron and a second one interrupting the acceptor domain of an overlapping external intron, both events leading to identical mature mRNAs. In the second case, an internal intron interrupts the donor domain of the external intron, while an alternatively spliced intron leads to an mRNA carrying a premature chain termination codon. We thus extend the stwintron concept to the acceptor domain and establish a link of the occurrence of stwintrons with that of alternative splicing.