Polyuridylylation and processing of transcripts from multiple gene minicircles in chloroplasts of the dinoflagellate Amphidinium carterae

Abstract

Although transcription and transcript processing in the chloroplasts of plants have been extensively characterised, the RNA metabolism of other chloroplast lineages across the eukaryotes remains poorly understood. In this paper, we use RT-PCR to study transcription and transcript processing in the chloroplasts of Amphidinium carterae, a model peridinin-containing dinoflagellate. These organisms have a highly unusual chloroplast genome, with genes located on multiple small 'minicircle' elements, and a number of idiosyncratic features of RNA metabolism including transcription via a rolling circle mechanism, and 3' terminal polyuridylylation of transcripts. We demonstrate that transcription occurs in A. carterae via a rolling circle mechanism, as previously shown in the dinoflagellate Heterocapsa, and present evidence for the production of both polycistronic and monocistronic transcripts from A. carterae minicircles, including several regions containing ORFs previously not known to be expressed. We demonstrate the presence of both polyuridylylated and non-polyuridylylated transcripts in A. carterae, and show that polycistronic transcripts can be terminally polyuridylylated. We present a model for RNA metabolism in dinoflagellate chloroplasts where long polycistronic precursors are processed to form mature transcripts. Terminal polyuridylylation may mark transcripts with the correct 3' end.

Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors

“…Characterizing the factors involved in polyuridylylation and editing will be a major task, as the effector proteins involved in chloroplast transcript editing and 3′ modification remain poorly characterized even in plants (20,40,41), and poly(U) polymerases involved in nuclear and mitochondrial RNA metabolism of other lineages appear to have arisen independently from a range of nucleotide polymerase families (41,42). In addition, it remains to be shown whether polyuridylylation and editing are functionally interconnected to other features of chloroplast RNA metabolism in K. mikimotoisuch as transcript end maturation-as has been hypothesized in peridinin dinoflagellate lineages (30,33,34).…”

“…For each reaction, 200 ng total cellular RNA was reverse-transcribed with SuperScript II reverse transcriptase (Invitrogen). For circular RT-PCR, RNA was precircularized using T4 RNA ligase as previously described (33,35). PCRs were performed using GoTaq polymerase (Promega) as previously described (33,35).…”

“…For circular RT-PCR, RNA was precircularized using T4 RNA ligase as previously described (33,35). PCRs were performed using GoTaq polymerase (Promega) as previously described (33,35). For the initial diagnostic oligo(dA) RT-PCR, PCR forward primers directed against the interior of each gene were used, whereas for whole-gene substitution mapping, PCR forward primers that annealed against the 5′ end of each gene were used (Dataset S1).…”

“…These include the addition of 3′ terminal poly(U) tracts, and (in some species) extensive transcript editing (3,(29)(30)(31)(32)(33). Outside the peridinin dinoflagellates, chloroplast RNA polyuridylylation has so far been reported only in the closely related species Chromera velia (29), and chloroplast transcript editing has been reported only in land plants (3).…”

Integration of plastids with their hosts: Lessons learned from dinoflagellates

“…Thus, the peridinin dinoflagellate plastid has lost all of the ancestral genes that would have encoded proteins of nonphotosynthetic function (18). There are a small number of genes that are not found in other plastid lineages and are specific to individual peridinin dinoflagellate species (29,30). It has additionally been suggested that the plastids of the peridinin dinoflagellates Ceratium horridum and Pyrocystis lunula may contain a small number of genes acquired through lateral transfers from bacterial sources although it cannot be excluded that these genes have been misidentified from bacterial contamination in the original sequence datasets (31).…”