Twelve Group I Introns in the Same Pre-rRNA Transcript of the Myxomycete Fuligo septica: RNA Processing and Evolution

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Abstract
The ribosomal DNA region of the myxomycete Fuligo septica was investigated and found to contain 12 group I introns (four in the small subunit and eight in the large subunit ribosomal RNAs). We have performed molecular and phylogenetic analyses to provide insight into intron structure and function, intron-host biology, and intron origin and evolution. The introns vary in size from 398 to 943 nt, all lacking detectable open reading frames. Secondary structure models revealed considerable structural diversity, but all, except one (subclass IB), represent the common group IC1 intron subclass. In vitro splicing analysis revealed that 10 of the 12 introns were able to self-splice as naked RNA, but all 12 introns were able to splice out from the precursor rRNA in vivo as evaluated by reverse transcription PCR analysis on total F. septica RNA. Furthermore, RNA processing analyses in vitro and in vivo showed that 10 of 12 introns perform hydrolytic cleavage at the 3′ splice site, as well as intron circularization. Full-length intron RNA circles were detected in vivo. The order of splicing was analyzed by a reverse transcription PCR approach on cellular RNA, but no strict order of intron excision could be detected. Phylogenetic analysis indicated that most Fuligo introns were distantly related to each other and were independently gained in ribosomal DNA during evolution.