Dinoflagellate spliced leader RNA genes display a variety of sequences and genomic arrangements

Abstract

Spliced leader (SL) trans-splicing is a common mRNA processing mechanism in dinoflagellates, in which a 22-nt sequence is transferred from the 5'-end of a small noncoding RNA, the SL RNA, to the 5'-end of mRNA molecules. Although the SL RNA gene was shown initially to be organized as tandem repeats with transcripts of 50-60 nt, shorter than most of their counterparts in other organisms, other gene organizations and transcript lengths were reported subsequently. To address the evolutionary gradient of gene organization complexity, we thoroughly examined transcript and gene organization of the SL RNA in a phylogenetically and ecologically diverse group of dinoflagellates representing four Orders. All these dinoflagellates possessed SL RNA transcripts of 50-60 nt, although in one species additional transcripts of up to 92 nt were also detected. At the genomic level, various combinations of SL RNA and 5S rRNA tandem gene arrays, including SL RNA-only, 5S rRNA-only, and mixed SL RNA-5S rRNA (SL-5S) clusters, were amplified by polymerase chain reaction for six dinoflagellates, containing intergenic spacers ranging from 88 bp to over 1.2 kb. Of these species, no SL-5S cluster was detected in Prorocentrum minimum, and only Karenia brevis showed the U6 small nuclear RNA gene associated with these mixed arrays. The 5S rRNA-only array was also found in three dinoflagellates, along with two SL-5S-adjacent arrangements found in two other species that could represent junctions. Two species contained multimeric SL exon repeats with no associated intron. These results suggest that 1) both the SL RNA tandem repeat and the SL-5S cluster genomic organizations are an "ancient" and widespread feature within the phylum of dinoflagellates and 2) rampant genomic duplication and recombination are ongoing independently in each dinoflagellate lineage, giving rise to the highly complex and diversified genomic arrangements of the SL RNA gene, while conserving the length and structure of the functional SL RNA.

FIG. 1.—

Dinoflagellate SL RNAs are 50–60 nt. Polyacrylamide gel electrophoresis of total RNA (A), and blot hybridization of probes for SL RNA using probe DinoSLa/s (B), and 5S rRNA using probe Dino5S (C). Lane 1, Karenia brevis; 2, Polarella glacialis; 3, Karlodinium veneficum; 4, Pfiesteria piscicida; 5, Prorocentrum minimum; and 6, Leishmania tarentolae. Size standards (marked on the left and the right) are L. tarentolae 5.8S rRNA (175 nt), 5S rRNA (110 nt) and tRNA^Gly (72 nt), and the RNA blots (B,C) were aligned with the gel so that the size standards apply for the RNA blots. The single band of K. veneficum SL RNA transcript (marked by an arrow) has been proven to be 56 nt in length by RACE-based cloning and sequencing (Zhang, Hou, et al. 2007).

FIG. 2.—

Alignment of dinoflagellate 5S ribosomal RNA gene using ClustalX. Promoter elements are indicated in Box A, I, and Box C. The region of dino5S probe binding is indicated by an arrow; positions where nucleotides differ from the probe are marked with shading. Har, Heterocapsa arctica; Kbr, Karenia brevis; Kve-1, Karlodinium veneficum, 5S in tandem repeats; Kve-2, K. veneficum, 5S clustered with SL RNA; Pgl, Polarella glacialis; Pmi, Prorocentrum minimum; Ppi-1, Pfiesteria piscicida, 5S in tandem repeats; and Ppi-2, P. piscicida, 5S clustered with SL RNA. “–” indicates missing nucleotides. Cco, Crypthecodinium cohnii (M25115); Pat (Perkinsus atlanticus, AF509333) are shown for reference.

FIG. 3.—

The complex genomic organizations of SL RNA genes in six dinoflagellates. Genes are oriented relative to the direction of the SL RNA gene when in tandem, and gene boxes below the line are transcribed from the opposite strand. Gray box, SL exon; open box, SL intron; vertically hatched box, 5S rRNA gene; diagonally hatched box, U6 snRNA gene; thin line, intergenic region; numbers near the lines or boxes depict length of sequence segments; unnumbered boxes are of the same length as immediately prior counterpart. *SL, structures reported previously (Zhang, Hou, et al. 2007); **, predicted intron sizes. Numbers in parentheses indicate the clones obtained for that type of SL RNA gene. Species are arranged so that basal taxa are on the bottom and later diverging taxa on the top, and their phylogenetic positions based on Zhang, Bhattacharya, and Lin (2007) are indicated in the clustering pattern shown on the left.

FIG. 4.—

Conservation of the ∼60-bp dinoflagellate SL RNA coding region (SL + intron). In the alignment are representative sequences for each type; the number of identical clones retrieved for each type is indicated by “@number” following the species abbreviation and type number. Har, Heterocapsa arctica; Kbr, Karenia brevis; Kve, Karlodinium veneficum; Ppi, Pfiesteria piscicida; Pgl, Polarella glacialis; and Pmi, Prorocentrum minimum. SL refers to SL RNA sequences obtained from SL-only repeats; SL–5S indicates SL RNA sequences from genes associated with 5S rRNA genes. *: Sequence from Zhang, Hou, et al. (2007); **: sequence from Lidie and van Dolah (2007). SL RNAs mapped by 3′-RACE analyses are denoted by arrows to indicate the terminal positions. The 22-nt SL (exon) was shown in uppercase letters, whereas the intron and the downstream region were in lowercase letters. Shaded are conserved positions defined as identical in over six sequences in at least three species. Where available, 5nt upstream of the 22-bp SL are shown for evaluation of a potential initiator element; sequences consistent with the initiator motif are underlined. Nucleotides existing in some clones but absent in others were italicized. A noncanonical C in the splice-donor site of KbrSL-3 is boxed. The conserved intergenic spacer region between different types of KbrSL are also boxed. Gaps introduced in the sequence alignment are shown as –.

FIG. 5.—

Predicted structures of SL RNA for Karenia brevis (A), Polarella glacialis (B), Heterocapsa arctica (C), and Pfiesteria piscicida (D). Model simulation was run using MFOLD: Prediction of RNA secondary structure modeling program (http://bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.html) under default setting. Simulation for different types of K. brevis SL RNA (KbSL) was based on isolated cDNA and that for P. glacialis (PglSL), H. arctica (HarSL), and the P. piscicida SL–5S form (PpiSL) was based on genomic sequences, by identifying conserved regions in the alignment of SL RNA genes with all the mapped dinoflagellate SL RNA transcripts. SL–5S denotes SL RNA transcribed from the SL–5S genomic cluster; SL-r denotes SL RNA transcribed from genomic tandem repeats of SL RNA gene. For more information, see text.