mRNA processing in Antonospora locustae spores

Abstract

Microsporidia are a group of intracellular parasites characterized by highly reduced and compact genomes. The presence of a high gene density had several consequences for microsporidian genomes, including a high frequency of overlap between transcripts of adjacent genes. This phenomenon is apparently widespread in microsporidia, and strongly correlated with gene density. However, all analyses to date have focused on one or a few transcripts from many loci, so it is unclear how diverse the pool of transcripts at a given locus may be. To address this question, we characterized initiation and termination points from 62 transcripts in gene-dense regions in Antonospora locustae spores using both conventional and fluorescence-based RACE-PCR procedures. In parallel, we investigated the abundance and nature of transcripts along a 6 kb region surrounding the actin locus of A. locustae using northern blotting, RACE-PCR and previously characterised EST sequences. Overall, we confirmed previous suggestions that most transcripts in A. locustae spores overlap with the downstream gene, but that at the 5' end untranslated regions are very short and overlap is rare. From fluorescence-based RACE-PCR we show that transcription of most genes (31 out of 34) initiates at a single position, whereas 35% of loci analyzed with 3' RACE polyadenylate mRNA at several sites. Finally, we identified the presence of previously unsuspected and very large transcripts in A. locustae spores. Those transcripts were found to overlap up to four open reading frames in different strands, adding a novel layer of complexity in the mRNA transcription of this microsporidian species.