Abstract
Because relatively little information is available about mtDNA in the euglenid protozoa, distant relatives of the kinetoplastid protozoa, we investigated mitochondrial genome structure and expression in Euglena gracilis. We found that isolated E. gracilis mtDNA comprises a heterodisperse collection of short molecules (modal size ~4 kbp) and that the mitochondrial large subunit (LSU) and small subunit (SSU) rRNAs are each split into two pieces. For the two halves of the SSU rRNA, we identified separate, non-contiguous coding modules that are flanked by a complex array of (primarily direct) A + T-rich repeats. The potential secondary structure of the bipartite SSU rRNA displays the expected conserved elements implicated in ribosome function. Label from [α-32P]GTP was incorporated in the presence of guanylyltransferase into each of the separate SSU and LSU rRNA fragments, confirming that these RNAs are primary transcripts, separately expressed from non-contiguous rRNA modules. In addition to authentic genes for SSU rRNA, we discovered numerous short fragments of protein-coding and rRNA genes dispersed throughout the E. gracilis mitochondrial genome. We propose that antisense transcripts of gene fragments of this type could have been the evolutionary precursors of the guide RNAs that mediate U insertion/deletion editing in the kinetoplastid relatives of the euglenids. To the extent that E. gracilis mtDNA is a representative euglenid mitochondrial genome, it differs radically in structure and organization from that of its kinetoplastid relatives, instead more closely resembling the mitochondrial genome of dinoflagellates in many of its features, an apparent evolutionary convergence.
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Acknowledgments
This work was supported by an operating grant (MOP-4124) from the Canadian Institutes of Health Research (CIHR) to M.W.G.
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Communicated by B. Franz Lang.
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Supplemental Figure R1.
Densitometry analysis to estimate the size of isolated Euglena mtDNA. Control and XbaI-cut Euglena mtDNAs were resolved on a 1% agarose, Tris-acetate gel. HindIII-cut λ DNA was included as a size marker. Densitometry of the ethidium bromide-stained was performed in ImageJ (Wayne Rasband, NIH) and the results are indicated beside the stained gel photo. From a calibration curve employing the λ DNA fragments, we estimate that the major fraction of uncut Euglena mtDNA is centered around 4 kbp while the minor, higher-molecular-weight fraction is centered around 7.5 kbp (as linear DNA). PDF 439 kb)
Supplemental Figure R2.
Photograph of agarose gel illustrating lack of detectable nucleolytic degradation of Euglena mitochondrial DNA preparations. Two fractions of Euglena mtDNA are shown: one fraction (lanes 1-7) clearly free of visible nuclear DNA as well the rRNA-encoding episome, the second (lanes 8-12) with small amounts of these contaminating DNAs present. The fractions were subjected to hydrolysis with various restriction endonucleases (cont = control untreated DNA) prior to gel electrophoresis. The higher-molecular-weight (genomic) DNA and the smaller 11.5 kbp episome serve as internal controls, supporting our conclusion that the observed condition of isolated Euglena mtDNA (heterodisperse collection of small molecules, most around 4 kbp in size) is not attributable to nuclease-mediated degradation. (PDF 2142 kb)
Supplemental Figure R3.
Northern hybridization analysis of Euglena mitochondrial RNAs. The left lane shows total Euglena mitochondrial RNA separated on a 6% denaturing polyacrylamide gel (4% crosslinking) and stained with ethidium bromide prior to capillary transfer to nylon membrane. The right-hand lanes are two exposures of the blot following hybridization with the ‘530 loop’ oligonucleotide that had been labeled at its 5′ end with [γ-32P]ATP and polynucleotide kinase. There is no evidence of any ‘full-length’ (i.e., spliced) small subunit rRNA (‘SSU’), which would be predicted to have a length of approximately 1100 nt. (PDF 738 kb)
Supplemental Figure 1.
Annotated E. gracilis mtDNA sequences. (A)XbaI fragment pEMX1-34 (rns-5′; GenBank acc. no. HQ266767); (B)XbaI fragment pEMX1-39 (rns-3′; GenBank acc.no. HQ266768); (C)XbaI fragment pEMX1-24 (no authentic genes; GenBank acc. no. HQ266766); (D)EcoRI fragment Y07965.1 (cox1) ; (E)EcoRI fragment AF156178.1 (cox2 + cox3). Arrows show the transcriptional orientations of genes and gene fragments and the relative orientations of flanking repeats. Upstream (UR) and downstream (DR) repeats are highlighted in yellow, with conserved inverted repeats underlined and color-coded in different shades of green. HindIII restriction sites within inverted repeats are shown as purple letters. Genes and corresponding gene fragments are highlighted in different colors: blue, 5′ coding region of the SSU rRNA (SSU-L); green, 3′ coding region of the SSU rRNA (SSU-R); red, cox1; orange; cox2; turquoise, cox3. Highlights in purple indicate blocks of LSU rRNA sequence. Gray highlight in (C) and (D) denotes non-coding sequences specifically conserved between these two clones. Translation initiation and termination codons are indicated by appropriately colored fonts on a white background. (PDF 3153 kb)
Supplemental Figure 2.
Proposed secondary structure of portions of the Euglena mitochondrial LSU-L and LSU-R rRNAs obtained by direct RNA sequencing. (A) GTPase domain within the 3′-terminal region of LSU-L. (B) α-Sarcin domain within 3′-terminal region of LSU-R. L5-1U, L5-1L, L3-1U and L3-1L denote the sequences of primers used in PCR amplifications (see text and Supplemental Fig. 4). (PDF 258 kb)
Supplemental Figure 3.
Alignment of upstream repeat, UR (A) and downstream repeat, DR (B) from XbaI clones pEMX1-24, pEMX1-34 and pEMX1-39. Identical residues are shown as white letters on a black background. Inverted repeats are indicated by colored underlining, with potential secondary structures shown on the right. ‘Egr Mt-L’ (A) and ‘Egr Mt-R’ (B) mark the sequences contained in PCR primers ‘L’ (5′-GTGATGAGGGTATCCTGAACGG -3′) and ‘R’ (5′-CATACAAAAACAATTAGGTATTGTAC-3′), respectively; see Supplemental Fig. 4. (PDF 222 kb)
Supplemental Figure 4.
Maps of six sequenced clones generated by PCR amplification employing primers to the most highly conserved regions of the UR (‘L’) and the DR (‘R’) (see Supplemental Fig. 3), a cox1 primer (‘cox9L’; 5′-CCRTATAATATACCTATTATYTTATG-3′), and four primers based on direct LSU rRNA sequence data and indicated in Supplemental Figure 2: L5-1U (5′-TCTAAGATTGATAGTATTCATGATA-3′); L5-1L (5′-TATCATGAATACTATCAATCCTAGA-3′), L3-1U (5′-GATTTATGTACGCAAGGATCAAATC-3′) and L3-1L (5′-GATTTGATCCTTGCGTACATAAATC-3′). The various repeats and identifiable gene fragments follow the coloring scheme employed in Figure 2 and Supplemental Figure 1, with larger colored terminal rectangles denoting the primer combinations employed. Clone designations are listed at the right of each map, with the length of each insert shown in parentheses. GenBank acc. nos. HQ266769 (L5-1U–R), HQ266770 (L3-1U–R(Top)), HQ266771 (L3-1U–R(Bottom)), HQ266772 (L–L5-1L), HQ266772 (L–L3-1L), HQ266774 (L3-1U–cox9L). (PDF 38 kb)
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Spencer, D.F., Gray, M.W. Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome. Mol Genet Genomics 285, 19–31 (2011). https://doi.org/10.1007/s00438-010-0585-9
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DOI: https://doi.org/10.1007/s00438-010-0585-9