Long transcripts from dinoflagellate chloroplast minicircles suggest "rolling circle" transcription

Abstract

The chloroplast genome of a dinoflagellate consists of a group of small circular DNA molecules (minicircles), most of which carry a single gene. With RT-PCR, primer extension, and Northern analyses, we show that the entire minicircle is transcribed and that some minicircles can produce RNAs larger than themselves. Using an RNA ligase-mediated rapid amplification of cDNA ends method, we were able to detect large processed precursors that are generated by endonucleolytic cleavage of an even longer molecule. This cleavage produces the mature mRNA 3'-end and at the same time the 5'-end of the precursor. The tRNAs encoded by the petD and psbE minicircles appear to be processed in the same way. We propose a "rolling circle" model for chloroplast transcription in which transcription would proceed continuously around the minicircular DNA to produce transcripts larger than the minicircle itself. These transcripts would be further processed into discrete mature mRNAs and tRNAs.

FIGURE 1.

Transcription of the psbB minicircle non-coding region tested with RT-PCR. Top, structure of psbB minicircle with the approximate positions of primers (arrowheads), open reading frame (heavy arrow), and non-coding region (NC, thin line). Bottom, RT-PCR results with different combinations of primers. The size marker is a 1-kb DNA ladder (Invitrogen). The cDNA was made with total RNA and random primers. Total RNA was used as control to test the residual DNA in the RNA sample. All PCR products were purified and sequenced.

FIGURE 2.

Detection of long minicircle transcripts. A, primer extension analyses of psbB, psbC, psaA, and psaB. For each sample, 30 μg of total RNA was used for cDNA synthesis with labeled gene-specific primers; reaction products were resolved on a 3.5% denaturing polyacrylamide gel containing 7 m urea. Interpretation of the results is shown beside the gel image. Table, lengths of minicircle, coding, and non-coding regions (in bp) and the names and positions of primers (first nucleotide of the start codon is +1). B, RNA analysis of psbB, psaA, psbC, and psaB minicircle transcripts. Total RNA was purified from cells growing in mid-light phase (L) or mid-dark phase (D). Total RNA (25 μg) was separated on a 1% formaldehyde agarose gel with Riboruler (Fermentas) as a size marker. The labeled probes for each gene (about 0.5 kb) were complementary to part of the protein-coding region.

FIGURE 3.

Determination of the 5′-end of atpA long transcripts. A, schematic of atpA minicircle and experimental strategy. Two sets of cDNA were made with the RLM-RACE method, using either T+ or T− total RNA. PCRs were carried out with various combinations of gene-specific reverse primers and forward primers complementary to the RNA linker (5′-RACE outer adaptor primer (5OP) and 5′-RACE inner adaptor primer (5IP)). Because the 5′-end of newly initiated RNAs carries triphosphates, it can only be detected in T+ samples. B, first and second round PCR. To detect the low abundant pre-RNAs, nesting PCRs were carried out with conditions shown below each lane. The first round PCR products (1st PCR) were diluted 200–500 times with distilled water and served as the templates for a second round of PCR (2nd PCR) with 5′-RACE inner adaptor primer and nesting reverse primers (atpAnr1, -2, or -3). All products were sequenced; correct products are outlined. The length of the long precursor determined by RLM-RACE is indicated by a thin arc in A. Size marker is a 1-kb DNA ladder (Invitrogen).

FIGURE 4.

Detecting the 3′-end cleavage site of the psbB minicircle transcript. The 5′-end of the long psbB precursor was determined by RLM-RACE as in Fig. 3. A, schematic of psbB minicircle with positions of primers used for 5′- and 3′-RACE (arrowheads). The black and grey arcs show the approximate span of 5′- and 3′-RACE (psbBf) relative to the DNA template. B, second round PCR with psbBnr2 and 5′-RACE inner adaptor (5IP) primers. The first round PCR used psbBr or psBnr1 primer. Both T+ and T− gave a single product. C, alignment showing the sequences of the long precursor RNA detected by RT-PCR (with psbBf and psbBr) compared with 3′-RACE and 5′-RACE results. The sequences of the poly(U) tail and the RNA linker (enzymatically added in 5′-RACE) are indicated by filled boxes. The open box indicates the stop codon of the psbB ORF.

FIGURE 5.

Detecting the 3′-end cleavage sites for trnW and trnP in petD minicircle transcripts with RLM-RACE. Left, schematic of petD minicircle and the approximate position of primers (arrowheads). The 5′-RACE was carried out as described in Fig. 3. First round PCR used petDr or petDnr1 primers; second round PCR used petDnr2 primer. Second round PCR results are shown in the gel image. The approximate spans of the 5′-UTRs for these two species of precursor RNA are shown in the outer and inner arcs (upper and lower gel bands, respectively). Right, partial alignment of 5′-RACE sequence with petD minicircle long transcripts detected by outwardly directed PCR as in Fig. 1. Open boxes, predicted tRNA genes (8); filled boxes, RNA linkers.

FIGURE 6.

Scheme describing the transcription mechanism of H. triquetra chloroplast minicircles. Possibly initiated randomly, transcription continues along the circular DNA template to produce long primary transcripts, which are subsequently cleaved by an endonuclease (possibly RNase Z) to generate pre-mRNA and/or pre-tRNA. Both precursors require further trimming at the 5′-end and other processing, such as 3′-polyuridylylation for mRNAs and 3′-CCA addition for tRNAs.