A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

Abstract

Edited by Ulf-Ingo FlüggeKeywords: Plastid EST Euglenozoa Polyadenylation Quantitative PCR Trans-splicing a b s t r a c t Euglena gracilis possesses secondary plastids of green algal origin. In this study, E. gracilis expressed sequence tags (ESTs) derived from polyA-selected mRNA were searched and several ESTs corresponding to plastid genes were found. PCR experiments failed to detect SL sequence at the 5 0 -end of any of these transcripts, suggesting plastid origin of these polyadenylated molecules. Quantitative PCR experiments confirmed that polyadenylation of transcripts occurs in the Euglena plastids. Such transcripts have been previously observed in primary plastids of plants and algae as low-abundance intermediates of transcript degradation. Our results suggest that a similar mechanism exists in secondary plastids. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. IntroductionEuglena gracilis is a fresh-water photosynthetic flagellate belonging to the order Euglenida and to the protist phylum Euglenozoa [1,2]. This phylum includes also the orders Kinetoplastida, Diplonemida and Symbiontida [3,4], which comprise exclusively heterotrophic species. Most euglenid species are free-living heterotrophic flagellates, but some of them possess secondary plastids that arose via secondary endosymbiosis of a green alga [5]. Phylogenetic analysis of plastid genome sequences revealed that euglenid plastids are derived from a Pyramimonas-related prasinophyte alga [6].A common euglenozoan feature is the processing of primary nuclear transcripts by spliced leader (SL) RNA-mediated trans-splicing [7]. This process includes replacement of the 5 0 -end of premRNA by the 5 0 -end of SL-RNA, donating identical 5 0 -termini to the mRNA molecules. Similar to nuclear cis-splicing, trans-splicing process is also mediated by spliceosomes, but a Y-branch intron structure is formed instead of a lariat [8]. The only currently known nuclear mRNA lacking the SL sequence in E. gracilis is that of the fibrillarin gene [9]. Since SL-trans-splicing does not occur in organelles (mitochondria, plastids), the presence (or absence) of an SL sequence at the 5 0 -end of a euglenozoan mRNA is diagnostic for its synthesis in the nucleus (or organelles, respectively).E. gracilis cell possesses approximately eight secondary plastids bounded by three membranes [10,11]. The plastid genome of this species is circular and comprises 143.17 kb. It contains 96 protein and RNA gene loci [12], group II and III introns, and twintrons (i.e. introns within introns) [13].The evolutionary transition from an endosymbiont to the plastid organelle was accompanied by a loss of many genes and gene transfer from the endosymbiont genome(s) to the host genome [14]. Gene transfer from plastids and mitochondria is an ongoing process, as it has been demonstrated in animals, plants, fungi as well as protists [15,16].Nuclear copies of plastid DNA (NUPT) can be found in a variety of species [17]. However, some species, e.g. the ...

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