Permuted tRNA Genes Expressed via a Circular RNA Intermediate in Cyanidioschyzon merolae

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Science  19 Oct 2007:
Vol. 318, Issue 5849, pp. 450-453
DOI: 10.1126/science.1145718

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A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3′ half of the tRNA lies upstream of the 5′ half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.

Cyanidioschyzon merolae is an ultrasmall unicellular red alga that inhabits an extreme environment (1). The complete sequence of the nuclear genome of C. merolae recently became available (2). Genome-wide analyses (1, 2) and a molecular phylogenetic analysis (3) have demonstrated that this organism is likely to represent one of the most ancestral forms of eukaryote. A search for tRNA genes from the C. merolae nuclear genome, using the tRNAscan-SE program (4), predicted only 30 tRNA genes encoding 30 species of anticodon, a number that is insufficient to decode all 61 codons (2). This prominent paucity of tRNA genes prompted us to search for undiscovered tRNA genes that may elucidate the evolution of tRNAs in early eukaryotes.

To search for C. merolae nuclear tRNA genes, we used SPLITS and SPLITSX, new programs that can detect cis-spliced tRNAs containing introns in various positions and trans-spliced tRNAs that are joined at several positions (57). In addition to this analysis, we performed a BLAST search of tRNA genes with conserved sequences in the TΨC arm or the anticodon arm, followed by manual inspection of the results.

The most important finding was that 11 genes have a novel gene organization in which the 3′ half of the tRNA sequence lies upstream of the 5′ half in the genome (Fig. 1A and fig. S1). Such a gene arrangement is accomplished by circular gene permutation (8), and we therefore termed these genes permuted tRNA genes. As shown in fig. S1, a TATA-like sequence is found upstream of the 3′ half in most of the genes. This sequence is also conserved in nonpermuted tRNA genes of the C. merolae nuclear genome. Thus, instead of the intragenic bipartite promoter consisting of an A box and a B box, which are conserved sequences in the D arm and TΨCarm (9), the upstream TATA-like sequence may play a central role in initiation of transcription in C. merolae. The genomic sequence encoding the intervening sequence between the 3′ and 5′ halves varies from 7 to 74 base pairs (bp). Downstream of the 5′ half, a T stretch that corresponds to a termination signal for RNA polymerase III (pol III) is found (10). These observations suggest that the pair of putative tRNA halves is transcribed as a linear RNA. As shown in Fig. 1B and fig. S2, permuted tRNA genes can be classified into four types on the basis of the location of the junction between the 3′ end of the 5′ half and the 5′ end of the 3′ half in the inferred secondary structures of the tRNAs. The junctions are located at 20/21 (between position 20 and 21) in the D loop (I), 37/38 in the anticodon loop (II), 50/51 in the TΨC stem (III), and 59/60 in the TΨC loop (IV). We identified one, six, one, and three candidates for the type I to IV tRNAs, respectively. Notably, the sequences adjacent to those junctions in tRNA precursors (pre-tRNAs) potentially form bulge-helix-bulge (BHB) motifs, which were originally found around the intron-exon junctions of nuclear and archaeal tRNAs (11).

Fig. 1.

Permuted tRNA genes in C. merolae. (A) Schematic representation of a permuted tRNA gene. (B) Inferred secondary structures for pre-tRNAs of four types of permuted tRNA genes. Arrowheads indicate the positions to be processed. The intron sequence is shown in lower case. The numbering of the tRNA positions is according to (19). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; G, Gly; K, Lys; L, Leu; M, Met; Q, Gln; R, Arg; and T, Thr.

Northern blot analysis using C. merolae total RNA verified that permuted tRNA genes are indeed transcribed in the cell (fig. S3). Next, to examine whether these tRNAs are aminoacylated, we performed Northern blot analysis of acid-urea gels with use of total RNA prepared from C. merolae cells under acidic conditions. Putative aminoacylated forms, which migrate more slowly than the deacylated forms, were detected, showing that these tRNAs could be aminoacylated in vivo (Fig. 2, lanes 1 and 2, and fig. S4). An in vitro aminoacylation analysis showed that recombinant C. merolae methionyl-tRNA synthetase (MetRS) methionylates tRNAiMet in total RNA preparations (Fig. 2, lanes 3 to 5). An in vitro transcribed tRNAiMet with the 3′ terminal CCA sequence was methionylated, although with less efficiency (Fig. 2, lanes 6 to 9). Thus, tRNA molecules expressed from permuted tRNA genes are aminoacylated and are likely to participate in protein synthesis.

Fig. 2.

Aminoacylation analysis of tRNAiMet(CAU). Total C. merolae RNA prepared under acidic conditions was separated on an acid-urea gel directly (lane 1) or after deacylation (lane 2). In vitro methionylation reaction mixtures were loaded onto the gel (lanes 3 to 9). Deacylated total RNA (lanes 3 to 5) or in vitro transcribed tRNAiMet with [wild type (WT), lanes 6 and 7] or without (ΔCCA, lanes 8 and 9) the CCA sequence was used.

What is the processing mechanism of the pre-tRNA for these unusual tRNA genes? To clarify the processing pathway, we detected processing intermediates by reverse transcription polymerase chain reaction (RT-PCR) with two different sets of primers (Fig. 3A), followed by sequencing analysis of those RT-PCR products (Fig. 3B and fig. S5). Analysis of the tRNAGln(CUG) verified the sequences of the pre-tRNAGln with a circularly permuted structure in which the leader sequence, the 3′ half, the intervening sequence, the 5′ half, and the trailer sequence were aligned in this order (Fig. 3B, lane 1, and fig. S5A). Interestingly, we also detected a circular RNA intermediate in which the leader and trailer sequences were removed and the resulting ends were ligated, while the intervening sequence was retained (Fig. 3B, lane 2, and fig. S5B). The existence of the circular RNA intermediate was confirmed by the generation of a PCR product representing two rounds of reverse transcription around the circular RNA (Fig. 3B, lane 2). The 3′-terminal CCA sequence is added posttranscriptionally in eukaryotes (12). To determine the terminal sequence of the mature tRNAGln, we performed RT-PCR with total RNA circularized by T4 RNA ligase. The sequence of the mature tRNAGln, in which the extra sequences are removed and the CCA sequence is added to the 3′ terminus of the acceptor stem, was verified (Fig. 3B, lane 3, and fig. S5C). As summarized in the model presented in Fig. 3C, maturation of the pre-tRNA probably starts with processing of the leader and trailer sequences, resulting in formation of the circular RNA intermediate. This processing step is most likely carried out by the tRNA-splicing machinery because the sequences adjoining the processing sites potentially form a BHB motif, which is the dominant recognition element for nuclear and archaeal tRNA-splicing endonucleases (11). The intervening sequence is then removed, possibly by ribonuclease (RNase) Pand tRNase Z (13, 14), followed by the CCA addition, to generate the correct termini. This model would be common to the permuted tRNA genes, because the circular RNA intermediate was detected for all 11 genes.

Fig. 3.

RT-PCR amplification of tRNAGln(CUG) and a model for the maturation of the permuted pre-tRNAs. (A) 5′ and 3′ primers used for RT-PCR are indicated as solid and broken arrows, respectively. (B) PCR products amplified from cDNA of pre-tRNAGln and from cDNA generated from one (×1) or two (×2) rounds of reverse transcription around a circular RNA intermediate or a circularized mature tRNAGln are indicated. Lane M, DNA molecular weight markers (Φ×174 DNA-Hae III digest). (C) Maturation of the pre-tRNA starts with processing of a BHB motif (boxed) by the tRNA-splicing machinery, resulting in formation of a circular RNA intermediate. The intervening sequence is removed by RNase P and tRNase Z, then followed by the CCA addition.

How could permuted tRNA genes have arisen? Permuted noncoding RNA (ncRNA) genes have been reported for Trypanosoma mitochondrial small subunit (SSU) ribosomal RNA (rRNA) (15) and a bacterial transfer messenger RNA (tmRNA) (16) that function in a two-piece form, in contrast to the C. merolae tRNAs reported here that function in a one-piece form. The permuted rRNA and tmRNA genes are hypothesized to have arisen by a gene duplication that formed a tandem repeat, followed by the loss of the outer segment of each copy (15, 17). Even if a circular permutation occurred in tRNA genes, most of the resulting permuted genes would not be retained in the genome because of the failure of expression or the loss of functional structure of the RNA. In C. merolae, however, permuted tRNA genes might have persisted in the genome because of the use of the upstream promoter by the transcription system and processing of the circularly permuted pre-tRNA into a canonical tRNA molecule by the tRNA-splicing machinery. Considering that C. merolae is an early rooted eukaryote and that the BHB motifs would play a pivotal role in the tRNA processing, it is possible that the permuted tRNA genes might have developed via a common process with the split-tRNA genes of Nanoarchaeum equitans (18). Further investigation should provide a hint about how to evaluate the evolution of tRNA genes in the early eukaryote.

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