Spliced leader RNA trans-splicing in dinoflagellates

Abstract

Through the analysis of hundreds of full-length cDNAs from fifteen species representing all major orders of dinoflagellates, we demonstrate that nuclear-encoded mRNAs in all species, from ancestral to derived lineages, are trans-spliced with the addition of the 22-nt conserved spliced leader (SL), DCCGUAGCCAUUUUGGCUCAAG (D ‫؍‬ U, A, or G), to the 5 end. SL trans-splicing has been documented in a limited but diverse number of eukaryotes, in which this process makes it possible to translate polycistronically transcribed nuclear genes. In SL trans-splicing, SL-donor transcripts (SL RNAs) contain two functional domains: an exon that provides the SL for mRNA and an intron that contains a spliceosomal (Sm) binding site. In dinoflagellates, SL RNAs are unusually short at 50 -60 nt, with a conserved Sm binding motif (AUUUUGG) located in the SL (exon) rather than the intron. The initiation nucleotide is predominantly U or A, an unusual feature that may affect capping, and hence the translation and stability of the recipient mRNA. The core SL element was found in mRNAs coding for a diverse array of proteins. Among the transcripts characterized were three homologs of Sm-complex subunits, indicating that the role of the Sm binding site is conserved, even if the location on the SL is not. Because association with an Sm-complex often signals nuclear import for U-rich small nuclear RNAs, it is unclear how this Sm binding site remains on mature mRNAs without impeding cytosolic localization or translation of the latter.inoflagellates are unicellular eukaryotes that contribute significantly to marine primary production, coral reef growth, and marine toxins. They are members of the Alveolata, which also include ciliates and apicomplexa (1). Dinoflagellate genomes are enormous (3-200 pg of DNA per cell) and lack typical histones, with chromosomes permanently condensed, nuclear membranes remaining intact in mitosis, and mitotic spindle being extranuclear (for review see ref.2). The mechanism of gene regulation is largely unknown. Sporadic investigations have shown that a relatively small fraction of genes are under transcriptional control (3-6) and that introns are not common (7,8). The few comprehensive studies of dinoflagellate gene structures reveal genes with high copy number and arrangement in polycistronic or otherwise tandem arrays (e.g., refs. 7-10).Spliced leader (SL) trans-splicing has been found in a disjointed group of eukaryotes, in which a short RNA fragment (i.e., SL, Ϸ15-50 nt) from a small noncoding RNA (SL RNA) is transplanted to the 5Ј end of independently transcribed pre-mRNAs to yield mature mRNAs. This process converts a polycistronic transcript into translatable monocistronic mRNAs. SL trans-splicing has been well studied in Euglenozoa. It has been detected in nematodes, Platyhelminthes, cnidarians, rotifers, ascidians, and appendicularia (for review, see 11-13). SL RNA contains two functional domains: an exon (i.e., SL) that is spliced to an mRNA and an intron that contains a spliceosomal (Sm) binding site be...

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates

“…1). The most basal lineage of ''core'' dinoflagellates was shown to be the genus Amphidinium, as has been found previously (Jørgensen et al, 2004;Zhang et al, 2007;Orr et al, 2012). The species Gymnodinium catenatum was clearly part of the Gymnodinium sensu stricto clade (100/1.00).…”

“…For example, as we have shown here, gene duplication and loss appears to be very common, and genes are presented in many copies in the genome, including pseudogenes, due to processes such as the ''recycling'' of processed cDNAs, in which they are reverse transcribed into the genome (Zhang et al, 2007;Slamovits and Keeling, 2008). The selfish operon model (Lawrence, 1999), postulated that gene clusters encoding a particular function were likely to stay intact over time, due to common selective processes operating on their function.…”

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

“…Transcription in dinoflagellates is poorly understood, and the discovery of a transspliced leader at the 5′ end of all dinoflagellate transcripts (11), in conjunction with the unusual tandem arrangement of gene copies and the lack of recognizable promoter elements in the intergenic spacer (9), has led to the proposal that dinoflagellates may synthesize long polycistronic transcripts (22). In this model, mature transcripts from TAGs have a single origin of transcription and the individual ORFs are excised by transsplicing at their 5′ end and by cleavage followed by polyadenylation at the 3′ end.…”

“…Interestingly, the spacer regions between the coding sequence of the tandemarranged genes have no recognizable transcription factor-binding motifs, confounding attempts to understand how gene expression is regulated. This observation, in concert with the discovery of transsplicing in dinoflagellates, has led to the proposal that dinoflagellate transcripts are polycistronic (11). This proposal is largely derived from studies in kinetoplastids, in which transsplicing and polyadenylation are used to excise ORFs from polycistronic transcripts (12).…”

Unfolding the secrets of coral–algal symbiosis

“…Dinoflagellates are characterized by a very large genome (Hackett et al, 2004) and a number of unique features such as DNA containing 5-hydoxymethylmuracil (Rae, 1976), a lack of the usual histones (Rizzo, 1981) and transcriptional regulatory elements (Li and Hastings, 1998). Dinoflagellates contain a conserved spliced leader sequence (Zhang et al, 2007), and highly expressed genes with elevated copy numbers and tandem repeats (Bachvaroff and Place, 2008). A number of dinoflagellate genes have been acquired from bacteria and other eukaryotes by horizontal gene transfer or endosymbiosis, resulting in important gene innovations (Wisecaver and Hackett, 2011;Wisecaver et al, 2013).…”