Multiple physical forms of excised group II intron RNAs in wheat mitoc
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《核酸研究医学期刊》
Biology Department, University of Ottawa Ottawa, Canada K1N 6N5
*To whom correspondence should be addressed. Tel: +1 613 562 5800 ext. 6356; Fax: +1 613 562 5486; Email: lbonen@science.uottawa.ca
ABSTRACT
Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3'terminaladenosines, as well as heterogeneous circular introns, which lack 3' nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.
INTRODUCTION
Almost all plant mitochondrial introns belong to the highly structured ribozymic group II class and the majority are located within NADH dehydrogenase (nad) genes encoding complex I components of the respiratory chain. To date, a total of 25 have been identified among various flowering plants, and that includes six of the trans-splicing type . Group II introns are also found in organellar genes of other eukaryotes (excluding animals), as well as in a few bacterial genes . In some cases, these introns have autocatalytic activity in vitro, even though intron-encoded RNA maturases and/or host-encoded protein machinery is required for splicing in vivo. Only a few examples of self-splicing introns have been reported for plant organelles, namely within algal chloroplast genes (5,6), but notably none in plant mitochondria.
Conventional group II introns are excised through two transesterification steps, with the 2'-OH of an unpaired adenosine 7–8 nt from the 3' end of the intron acting as the first attacking nucleophile. This is the same biochemical mechanism as for nuclear spliceosomal introns and such similarities suggest that they share a common evolutionary origin . For both classes, the intron is liberated as a lariat, although group II ones have a much shorter tail and are highly structured with six distinctive domains (dI-dVI). From detailed biochemical and mutational analysis (primarily of fungal mitochondrial and bacterial ribozymic introns), key interactions required for proper folding and autocatalytic activity in vitro have been elucidated, and they include dV as part of the catalytic core and dVI which provides the first attacking nucleophile . In particular, branching requires an adenosine in a bulged context within the dVI helix and at a fixed distance from the 3' splice site, namely 7 nt for group IIA introns and 8 nt for group IIB (3,8–10). Mutations within the dV/dVI region, particularly at the branchpoint, can shift the first splicing step from transesterification to hydrolysis, and in some cases to incorrect selection of the 3' splice site (11,12).
In contrast to most other group II introns examined to date, those in plant mitochondria do not always conform to the conventional group II secondary structural model . The core helices can have weak base-pairing and certain key features sometimes appear missing. This raises questions about the mechanistic and evolutionary pathways taken by such ‘degenerate’ introns. Interestingly, a plant chloroplast intron, trnV, which lacks a dVI bulged adenosine, is excised as a linear molecule which is generated by a hydrolytic pathway of splicing (13). The importance of the dVI structure for efficient splicing in plant mitochondria is also supported by mutational analysis of the wheat cox2 intron, in that disruption of base pairing resulted in loss of splicing as monitored by in organello splicing assays (14). In the present study, we have examined the excised physical forms of the wheat mitochondrial introns nad2 intron 4, nad1 intron 2 and cox2 intron 1, designated as nad2i1282, nad1i477 and cox2i373, respectively in the nomenclature of Dombrovska and Qiu . We provide evidence for multiple splicing pathways in wheat mitochondria.
MATERIALS AND METHODS
RT–PCR and cRT–PCR analysis of intron RNAs
Mitochondrial RNA was isolated from 24 h germinating wheat embryos (Triticum aestivum cv. Frederick) as previously described (16). To examine the sequences of lariat (or circularized) excised intron molecules, a synthetic oligomer mapping close to the 5' end of the intron was used as primer in cDNA synthesis with Superscript II reverse transcriptase (Invitrogen), which is able to cross 2'–5' bonds (17). Wheat mitochondrial RNA (5 μg) was denatured at 70°C for 10 min and cDNA was synthesized using 10 U/μl reverse transcriptase, 0.2 vol 5x First Strand buffer (Invitrogen), 10 mM DTT and 0.5 mM dNTPs incubated at 45°C for 2 h. Subsequently, this primer in combination with one mapping to the 3' end of the same intron was used in PCR amplification with Taq DNA polymerase (Invitrogen). In parallel, cRT–PCR experiments (to circularize any linear intron molecules which contain a 5' monophosphate) were performed (18) with wheat mitochondrial RNA (5 μg) being incubated with 0.6 U/μl RNA ligase (New England Biolabs), 0.5 U/μl RNasin (Promega), 50 μg/ml BSA (Promega) and 0.1 vol 10x RNA ligase buffer (New England Biolabs) overnight at 14°C, and then recovered by ethanol precipitation prior to PCR amplification. The resulting cRT–PCR products will also include any endogenously circularized molecules already present in the RNA population. Prior treatment of RNA by heating at 70°C for 10 min or boiling for 5 min did not affect the nature or yield of products. In some experiments, Superscript III reverse transcriptase (Invitrogen) was used, although it appeared less efficient in traversing 2'–5' bonds. In the RT–PCR experiments, no product was seen in the absence of RT or when DNA was used as template with the cRT–PCR primer pairs (because of their inverted orientation). The sole exception was an abundant low molecular weight product for nad1 intron 2, which was determined to be co-linear with DNA by sequence analysis.
RT–PCR or cRT–PCR products were subsequently gel-purified using UltraClean 15 (MoBio Laboratories Inc.) and either sequenced directly using Sequenase version 2.0 (US Biochemicals) and electrophoresed on urea-polyacrylamide sequencing gels or after being cloned into pGemT-Easy (Promega) plasmid vectors (19). Automated sequencing was performed by the Ottawa Health Research Institute DNA sequencing facility (OHRI) and the McGill University/Genome Québec Centre.
RNase H analysis of excised introns
Wheat mitochondrial RNA (5 μg) was incubated with intron-specific oligomers at 37°C for 30 min prior to treatment with 0.03 U/μl RNase H (Invitrogen), 1 U/μl RNasin (Promega), 0.27 μg/μl BSA (Promega) and 10 mM DTT at 37°C for 30 min (20). RNase H-treated samples were phenol-extracted and ethanol-precipitated prior to northern blot analysis. Hybridizations were performed with 32P-end-labelled (T4 polynucleotide kinase) oligomer probes designed based on wheat mitochondrial sequences. The synthetic oligonucleotides used for RT–PCR, cRT–PCR, RNase H and as hybridization probes are shown in Supplementary data (online).
RESULTS
Nature of dVI branchpoint region of plant mitochondrial group II introns
Most plant mitochondrial introns fall in the group IIA category, in which the bulged adenosine of dVI is located 7 nt from the 3'end of the intron (Figure 1A, boxed) and the two unpaired terminal nucleotides (5'-AY-3') are involved in long-range interactions including –' with the dII/III linker . Figure 1B shows such an example in nad2 intron 4, which conforms to this conventional structure and its sequence is highly conserved among plants (Figure 1B, inset box). In contrast, a subset of plant mitochondrial introns, of both cis- and trans-splicing types, deviate from the classical group II structure to varying extents (Figure 1C–H). The dVI helices of some have a lower predicted thermodynamic stability than for classical group II introns, and this can be seen by A:G mispairs in the cox2 intron (Figure 1C), adjacent A:A and G:G mispairs in nad5 intron 2 (Figure 1D), A:A mispairs in nad4 intron 2 (Figure 1F) and to a much more pronounced extent in nad1 intron 1 . It is notable that plant mitochondrial intron folding can be improved by RNA editing, which converts A–C mis-pairs to A–U pairs, although not all such mispairs in dV/dVI regions are seen to undergo editing in the introns of mitochondria (23) or chloroplasts (13), and very few of the dVI mispairs shown in Figure 1 fall in that category (Figure 1D and H, but cf. underlined lower case U in dVI of nad5 intron 2). Even those introns possessing strongly paired dVI helices, in several cases deviate from the conventional group II format of having a purine stretch opposite a string of downstream pyrimidines (Figure 1D–E, G–H). The cox2 intron contains an A:G mispair immediately distal to the dVI branchpoint, as do a number of barley chloroplast introns, yet all of those were found to be excised as conventional lariats (13).
Figure 1 Secondary structure models of domains V and VI of wheat mitochondrial group II introns. (A) Classical group IIA intron (boxed), (B) nad2 intron 4 (Y14434), (C) cox2 intron (X01108), (D) trans-splicing nad5 intron 2 (M74158), (E) nad2 intron 1 (Y14433), (F) nad4 intron 2 (X57164), (G) nad1 intron 2 (X57967) and (H) trans-splicing nad1 intron 1 (X57967). The introns shown in (B–H) are designated as nad2i1282, cox2i373, nad5i1455, nad2i156, nad4i976, nad1i477 and nad1i394, respectively in the nomenclature of Dombrovska and Qiu . Expected position of dVI bulged adenosine is indicated by an asterisk. Non-conserved positions are shaded and boxes below each wheat intron (AP008982) show homologous regions from the sequenced mitochondrial genomes of rice (Oryza sativa BA000029), maize (Zea mays AY506529), Arabidopsis thaliana (NC_001284), Brassica napus (AP006444), tobacco (Nicotiana tabacum NC_006581), and sugar beet (Beta vulgaris NC_002511), and selected introns from Oenothera berteriana (M81725, M63033) and Pisum sativum (22). Dots represent identical nucleotides and lower case underlined nucleotides show C-to-U editing and differ from Y14434 and X57164, respectively.
With respect to the lariat-generating branchpoint, the majority of plant mitochondrial introns possess a bulged adenosine at the expected position, however there are several interesting exceptions (Figure 1E–H). For example, a bulged nucleotide is completely absent from the dVI helix of nad1 intron 2 and its short tight structure is conserved among all flowering plants examined to date. In contrast, in the very weakly structured dVI of nad1 intron 1, the first candidate adenosine is located at least 11 nt from the 3' end (Figure 1H) and considerable variation in sequence is seen among plants. In the case of nad2 intron 1 and nad4 intron 2 (Figure 1E–F), although a bulged nucleotide is present at the expected location (albeit flanked by pairs of varying strength among plants) it is not adenosine (cf. nad4 intron 2) or adenosine in only some plants (cf. nad2 intron 1).
Indeed the extent of sequence variation among orthologous introns from various plants in the vicinity of the branchpoint and its impact on potential base pairing (Figure 1E, F and H, shaded residues and insets) is surprising given the crucial role of dVI in lariat formation, as well as the low rate of nucleotide substitution in plant mitochondrial DNA (24). This is not the behaviour expected of sequences under strong functional constraint. In this regard, we had previously noted that dV helices also tend to show weaker pairing than classical group II introns (23). In some cases, dV structure is improved by editing, but in other instances the mispairs either remain unedited or they are not editing candidates (cf. Figure 1D and F, boxed Cs and cox2 intron U:U). Incidentally, nad4 intron 2 and nad1 intron 1 also have aberrantly long dV loops, and this is correlated with weak dVI helices and high sequence variation among plants (Figure1F and H). Based on such examination of dVI features, we selected three cis-splicing introns from wheat for this study, namely, nad2 intron 4 which has a conventional dVI (Figure 1B), nad1 intron 2, which has a short tight dVI helix (Figure 1G), and the cox2 intron, which has an A:G mispair adjacent to the branchpoint and mispairing in dV (Figure 1C).
Atypical circularized and linear introns detected by RT–PCR and cRT–PCR
To assess the presence of lariat (and/or circularized) excised introns in wheat embryo RNA populations in vivo, we used an RT–PCR strategy to amplify and then subsequently sequence the junction regions of nad2 intron 4, nad1 intron 2 and the cox2 intron. These experiments were accompanied in parallel with RNA ligase-treated samples (cRT–PCR) to determine whether linear intron molecules might also be present in the population (Figure 2). We included rps7 mRNA as a control, as it is known to be a 5' processed transcript (25) and thus can be circularized with RNA ligase. As expected, the rps7 mRNA template resulted in an RT–PCR product of 450 bp only when pre-treated with RNA ligase (Figure 2A, lane 1 versus lane 2, arrowhead).
Figure 2 RT–PCR and cRT–PCR detection of lariat (and/or circularized) versus linear wheat mitochondrial intron molecules. (A) RT–PCR across the excised intron junction using 24 h wheat mitochondrial RNA as template, either pre-treated with ligase (+L; lanes 2, 5, 7 and 9) or untreated (–L; lanes 1, 4, 6 and 8) generated products of sizes 450, 520, 630 and 400 bp (denoted by arrowheads) for rps7 mRNA, nad2 intron 4, cox2 intron and nad1 intron 2, respectively. Size markers are shown in lane 3. (B) Southern blot of gels (shown in A, lanes 4–9) using intron-specific oligomer probes for nad2 intron 4, cox2 intron and nad1 intron 2. (C) Schematic showing positions of primers (arrows 1 and 2) used for RT–PCR and cRT–PCR in panels (A) and (B). Branchpoint is shown by A. Dotted lines indicate regions of amplification. (D–F) Direct sequencing of RT–PCR products for (D) cox2 intron (+L), (E) nad1 intron 2 (–L) and (F) nad1 intron 2 (+L) using oligomers 6, 11 and 16, respectively (Supplementary data). Note that an inverted orientation of the sequencing gel is shown for (E). Arrows show positions of the 5' terminal nucleotide of the introns, stars highlight short non-encoded A-rich stretches and the discrete non-encoded insert sequence is boxed in (E).
For nad2 intron 4, nad1 intron 2 and the cox2 intron, we observed RT–PCR products for both the RNA ligase-treated and untreated templates, and their respective sizes were as expected for excised introns (Figure 2A, lanes 4–9, arrowheads). Several larger products were also observed (Figure 2A, lanes 4, 6, 7); however only nad2 intron 4 (Figure 2A, lane 4) exhibited discrete hybridization signals with intron-specific probes in Southern blot analysis (Figure 2B, lane 4). The latter may reflect amplification products which include neighbouring nad2 sequences, whereas those generated with the cox2 intron primers appear to be due to mis-priming from an unrelated genomic region. Similarly, higher molecular weight products were reported in the analysis of barley chloroplast introns (13). The apparent shift from discrete to heterogeneous high molecular weight products when RNA ligase-treated template was used with nad2 intron 4 primers (Figure 2A, lane 4 versus lane 5) may relate to contribution from circularized nad2 trans-splicing precursor molecules. In the case of nad1 intron 2, the abundant low molecular weight species (Figure 2A lanes 8, 9) were confirmed by sequence analysis to be mis-primed products containing an internal section of nad1 intron 2 that is co-linear with mitochondrial DNA (Figure 2B, lanes 8, 9), and this is consistent with the absence of Southern hybridization signal because of the intron oligomer probe maps outside that region. Comparable results with those shown in Figure 2 were reproducibly obtained with templates from different mitochondrial RNA preparations, different cDNA preparations and when alternative primers were used for cDNA synthesis.
To determine the precise nature of the excised intron junction, these RT–PCR products were cloned and sequenced. Such data are shown in Table 1. As expected, the nad2 intron 4 junction sequence was consistent with the excised intron being a lariat (with a 6 nt tail) based on six out of six RT–PCR clones and four out of four cRT–PCR clones. At the branchpoint position, we observed mis-incorporation of adenosine instead of thymidine, as has been previously reported for templates with 2'–5' bonds (13,17,22).
Table 1 Sequence analysis of RNA-ligase treated (+L) and untreated (–L) RT–PCR clones of circularized or lariat intron junction regions for wheat mitochondrial nad2 intron 4, the cox2 intron and nad1 intron 2
In contrast, for the cox2 intron only one out of five RT–PCR clones was consistent with a lariat model of splicing (Table 1). This was unexpected as this intron contains a bulged A in dVI (Figure 1B). The other four clones displayed heterogeneous circles each missing different lengths (from 10–29 nts) from the 3' terminal region of the intron. It should be noted that 50% of the clones obtained from multiple experiments had the correct insert size but sequencing revealed heterogeneous stretches of upstream and/or downstream exon sequences in addition to all or part of the intron (data not shown). This was not seen in our examination of any of the other introns. Such species might be derived from degraded RNA molecules, yet it is curious that they appear to be endogenously circularized. In this regard, evidence has been presented for a potential cryptic 5' splice site within cox2 exon 1 of petunia (26), but that appeared to be discrete rather than heterogeneous in nature.
When the cox2 intron cRT–PCR (RNA ligase-treated) products were cloned and sequenced to assess the presence of any linear intron RNA species in the population, we found that six out of eight clones had short non-encoded polyA tracts (1–5 nt in length) and four of these were full-length introns whereas the other two lacked 3' nucleotide stretches. In addition, one clone represented a full-length circularized intron species (end-to-end) and one simply lacked 3'terminal nucleotides. The latter presumably reflects contribution from the pre-existing non-RNA-ligase treated population of endogenously circularized forms. Notably in all cases, the precise 5' end of the cox2 intron was observed. The presence of a mixed population of excised cox2 intron species was corroborated by direct sequencing of the cRT–PCR products (Figure 2D). It can be seen that the sequence is homogeneous until the 5' end of the intron, but immediately past the junction there is a mixture of A-rich sequences (Figure 2D, asterisk).
The nad1 intron 2, which contains a short tight dVI helix, showed yet a different set of features in its population of excised intron molecules and none were lariats. Rather 5/9 clones showed a discrete 5'-ACAAAAC-3' insert at the junction of full-length introns, and its relative abundance is evident in the directly sequenced population of RT–PCR products (Figure 2E, boxed nts). Of the remaining four clones, one represented a full-length circularized intron, while two clones also contained 5'-ACTGT-3' which matches the first 5 nt of nad1 exon 2, and one clone lacked the 3' terminal 32 nt of the intron (Table 1). Similar results were observed for the cRT–PCR analysis, in that 3/10 clones showed precise end-to-end circularized species, and the others also contained either the non-encoded ACAAAAC block (4/10 clones) or ACTGT (3/10 clones). When the cRT–PCR products were examined directly, the sequence after the junction (Figure 2F) appears to be somewhat more heterogeneous than what was seen for the in vivo population (Figure 2E).
Linear and circular excised introns in vivo supported by RNase H analysis
Although the above strategy provides information about the physical form of excised introns, it does not directly address their relative abundance, especially since the yields of RT–PCR products may be influenced by reverse transcriptase being less efficient at extending over the 2'–5' bond of a lariat species compared with the 3'–5' bond generated by ligation of the ends of a previously linear molecule (cf. Figure 2C). To assess the relative levels of spliced lariat (and/or circularized) versus linear physical forms of introns in vivo, wheat mitochondrial RNA was annealed with intron-specific oligomers prior to incubation with RNase H and subsequent northern blot hybridization. In our analysis, we included nad7 intron 4 which we had previously shown to be predominantly lariat, but which also exhibited a minor population of fully circularized (end-to-end) intron molecules (16,27), and as expected, the vast majority appear to be lariat rather than linear (Figure 3A, black vs. open arrowhead). In the case of nad2 intron 4, when we used two annealing oligomers prior to RNase H treatment, again the shift in size was consistent with a lariat (or circularized) excised intron (Figure 3B, black arrowhead denoting 900 nt product detected by probe), when compared with the native 1.4 kb intron (Figure 3B, black dot). A minor signal was detected at the position corresponding to a linear form (400 nt, Figure 3B, open arrowhead). Also, major products of 4.8 kb derived from the linear precursor of 6 kb can be seen (Figure 3B) in addition to the precursor and intron species which were not completely digested by RNase H.
Figure 3 Determination of in vivo abundance of lariat (and/or circularized) versus linear excised introns by RNase H analysis. Wheat mitochondrial RNA (24 h) was annealed with oligomers as shown in schematics (arrows a–h) for (A) nad7 intron 4, (B) nad2 intron 4, (C) cox2 intron and (D) nad1 intron 2, prior to RNase H treatment. Positions of oligomer probes used in subsequent northern blot analysis are shown by overlined asterisks. Size markers are shown, black dots represent the positions of native excised introns, black arrowheads indicate positions expected for hybridizing products from lariat (or circularized) intron forms and white arrowheads for linear intron forms.
RNase H analysis of the cox2 intron using two intron-specific primers revealed conversion of the 1.1 kb intron (Figure 3C, black dot) to products consistent with circular (or lariat) molecules of 600 nt (Figure 3C, black arrowhead) and linear ones of 300 nt (Figure 3C, open arrowhead), as well as a shift in the 2.6 kb precursor to a prominent species of 800 nt. The relative strengths of the hybridization signals for the linear and circular forms (Figure 3C, open versus black arrowheads) suggest that both species are rather equally represented in the population, and this corroborates the results obtained with RT–PCR and cRT–PCR (Figure 2 and Table 1).
In the case of wheat nad1 intron 2, more complex northern profiles are observed because there are multiple precursors due to its co-transcription with upstream atp6 and rps13 genes, and this results in RNA species of 6, 4.6 and 3.6 kb (Figure 3D). After RNase H treatment, when only one intron-specific primer was used (oligomer g), the signal for the excised intron of 1.4 kb shifts to a 1.2 kb product consistent with the presence of a linear species (Figure 3D, black dot versus open arrowhead). This outcome was also seen when oligomer h was used, the shift being to 960 nt (Figure 3D, open arrowhead). In both cases, the linear species appear relatively abundant. Lariat (or circular) species when converted to Y-shaped structures by RNase H, would be expected to co-migrate with the native form under these electrophoretic conditions, but there is very little signal at that position (Figure 3D, black arrowhead and black dot). The high molecular weight precursors shift to the expected sizes of 2.6 and 2.3 kb. It is worth noting that the RNA ligase (cRT–PCR) experiments (cf. Figure 2A, lane 9) might be influenced by protective structures at the intron termini, such as the 3' terminal hairpin of nad1 intron 2.
DISCUSSION
Our analysis of the cox2 and nad1 excised introns in RNA isolated from germinating wheat embryos has uncovered novel physical forms which deviate from the classical lariat structure of group II introns. In contrast to introns such as nad2 intron 4 (this work), nad7 intron 3 (21,27) and nad4 intron 3 (N. Niknejad and L. Bonen unpublished data) which possess a bulged adenosine branchpoint in dVI and are excised as lariats, our observations in the case of nad1 intron 2 and the cox2 intron support the presence of both circularized and linear molecules in vivo (Figure 4). Because the cox2 intron has an apparently conventional branchpoint adenosine, it was unexpected that the lariat form was not predominant, but instead we observed heterogeneous circular excised introns which lack various lengths at the 3'terminus. Moreover, the population included full-length linear intron molecules with short, non-encoded polyA stretches.
Figure 4 Models for plant mitochondrial group II intron splicing. (A) Conventional two-step transesterification yielding intron lariat. Upstream and downstream exons are denoted by dark and light shaded boxes, respectively. (B) Hydrolytic pathway generating linear excised intron which can further undergo (i) 3' polyadenylation, (ii) 3' exonuclease activity prior to endogenous circularization of heterogeneous molecules, , (iii) addition of discrete non-encoded block (hatched box) prior to endogenous circularization or (iv) incorrect 3' splice site selection (dashed arrow) resulting in a discrete exon fragment (ACTGT for nad1 intron 2, light shaded box) being incorporated within the circular molecule. Note that the spliced exon–exon product is only shown in panel (A). (C) Hypothetical pathways of splicing via (i) external nucleophilic attack by a discrete non-encoded block (ACAAAAC, hatched box) followed by circularization of the full-length intron, (ii) an internal intronic nucleophile for first transesterification step (dashed arrows) or (iii) the hydroxyl group of the free upstream exon (dark shaded box) providing the first attacking nucleophile, followed by the terminal pyrimidine (Y) of the intron acting as the circularizing nucleophile .
Taken together, our data for the cox2 intron support a hydrolytic pathway of splicing generating linear molecules which undergo either (i) polyadenylation at the 3' terminus or (ii) endogenous circularization after 3' exonucleolytic attack (Figure 4B). Linear molecules have also previously been reported for the Streptococcus GBS intron, which possesses a conventional branchpoint adenosine (29), and for the barley chloroplast trnV intron, which in addition is polyadenylated (13); however, the wheat cox2 intron population includes endogenously circularized molecules as well. This might occur through nucleophilic attack by 2'-OH groups at other positions within dV or dVI to generate aberrant lariats with longer tails of varying lengths (Figure 4C). However, the cloned sequences correspond identically to the genomic sequence at such putative novel branchpoints, whereas typically an adenosine branchpoint is mis-read as T by reverse transcriptase (13,17,22). Alternatively, circularization might occur via an endogenous RNA ligase. It is also not excluded that the linear molecules which we observed are derived from the debranching of lariats with subsequent protection from exonuclease attack.
The nad1 intron 2 appears to be present as linear and circular molecules in vivo, but they have quite different physical features compared with the cox2 intron (Figure 4B and C). Notably, the excised introns fall into three categories: (i) precisely full-length molecules, (ii) those which in addition have a discrete non-encoded ACAAAAC block and (iii) ones which have the full-length intron plus ACTGT (which corresponds to the first 5 nt of the downstream exon). The latter could be derived from incorrect 3' splice site selection and such alternative splicing would lead to defective mRNA molecules. Because exactly the same non-encoded nucleotide blocks were consistently seen (and with or without RNA ligase treatment), these observations differ from those for the cox2 intron and do not easily conform to a model of being generated by a polyA ragged tailing mechanism (Figure 4B). The endogenous linear molecules, which comprise the majority based on RNase H analysis, appear resistant to 3' exonuclease attack (unlike the cox2 intron) and this may be related to the short tight dVI helix . In all cases, the correct 5' splice site was seen both for nad1 intron 2 as well as the heterogeneous cox2 intron molecules. The apparent absence of exonuclease attack at the 5' end of putative linear molecules might relate to protective RNA structures or close association with proteins.
Our nad1 intron 2 data suggest a combination of splicing mechanisms which exploit various internal or external nucleophiles to explain the discrete non-encoded block (Figure 4B and C, hatched box). The latter would be analogous to group I intron splicing where an external guanosine acts as the attacking nucleophile . The origin of the non-encoded ACAAAAC block is unknown. If it is added to the 3' end of the intron, it seems unlikely to be mediated by tRNA nucleotidyl transferase or an RNA-directed RNA polymerase, as was suggested for tobacco chloroplast ndhD mRNA processing (34), since there is no obvious template. Other models consistent with a precisely full-length circularized intron (which we observed at low levels for nad1 intron 2, nad7 intron 4 and the cox2 intron) include reverse splicing or a ‘spliced exon reopening’ pathway whereby the 3'-OH of the liberated 5'-exon acts as first attacking nucleophile in trans and then the hydroxyl group of the intron terminal pyrimidine attacks the 5' splice site (Figure 4C). Interestingly, in plant mitochondria, uncoupled splicing intermediates have been observed to be relatively abundant in some cases (16) and thus might contribute to such a pathway.
The apparent loss of classical group II features of mitochondrial introns in the flowering plant lineage over evolutionary time is supported by comparisons with homologues from early-diverging lineages of land plants. For example, nad1 intron 1 in Isoetes lacustris (22,36) and the cox2 intron in Acorus calamus (37) have conventional dVI structures. Moreover, the dynamic nature of plant mitochondrial genomes can have impact on intron sequences, such as the genomic rearrangements which have led to trans-splicing. It also appears that gene conversion (or swapping) events can occur between group II core structures. For example in Figure 1, it can be seen that dV and part of dVI of nad1 intron 2 and nad2 intron 1 are identical. This also underscores the plasticity and tolerance that the splicing machinery must have in light of such deviation from conventional structure.
The mixture of physical forms of excised introns observed in this study suggests that multiple biochemical mechanisms of splicing occur in plant mitochondria, such as a hydrolytic pathway as well as unusual circularization events. Their relative contributions appear to vary among introns and it will be interesting to learn if there are developmental effects, perhaps due to differences in the availability of nuclear-encoded machinery especially early in germination when seeds leave dormancy (16). In this regard, it is interesting that the steady state levels of excised introns show marked differences during germination, typically being present at higher levels in embryos than in seedlings. The apparently ‘degenerate’ nature of the folding of these group II introns raises the possibility that additional proteins and/or small RNAs have been recruited for the splicing process in plant mitochondria.
SUPPLEMENTARY DATA
Supplementary data are available at NAR Online.
ACKNOWLEDGEMENTS
The authors thank C. Carrillo, N. Niknejad, S. Subramanian and J. Crosthwait for their earlier work on wheat and rice mitochondrial introns in our laboratory. The authors also thank Dr R. Pandeya (Agriculture and Agri-Food Canada, Ottawa) for kindly providing the wheat seeds used in this study. Financial support in the form of a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (L.B.) and an Ontario Graduate Scholarship (J.L.P.T.) is gratefully acknowledged. Funding to pay the Open Access publication charges for this article was provided by the Natural Sciences and Engineering Research Council of Canada.
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*To whom correspondence should be addressed. Tel: +1 613 562 5800 ext. 6356; Fax: +1 613 562 5486; Email: lbonen@science.uottawa.ca
ABSTRACT
Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3'terminaladenosines, as well as heterogeneous circular introns, which lack 3' nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.
INTRODUCTION
Almost all plant mitochondrial introns belong to the highly structured ribozymic group II class and the majority are located within NADH dehydrogenase (nad) genes encoding complex I components of the respiratory chain. To date, a total of 25 have been identified among various flowering plants, and that includes six of the trans-splicing type . Group II introns are also found in organellar genes of other eukaryotes (excluding animals), as well as in a few bacterial genes . In some cases, these introns have autocatalytic activity in vitro, even though intron-encoded RNA maturases and/or host-encoded protein machinery is required for splicing in vivo. Only a few examples of self-splicing introns have been reported for plant organelles, namely within algal chloroplast genes (5,6), but notably none in plant mitochondria.
Conventional group II introns are excised through two transesterification steps, with the 2'-OH of an unpaired adenosine 7–8 nt from the 3' end of the intron acting as the first attacking nucleophile. This is the same biochemical mechanism as for nuclear spliceosomal introns and such similarities suggest that they share a common evolutionary origin . For both classes, the intron is liberated as a lariat, although group II ones have a much shorter tail and are highly structured with six distinctive domains (dI-dVI). From detailed biochemical and mutational analysis (primarily of fungal mitochondrial and bacterial ribozymic introns), key interactions required for proper folding and autocatalytic activity in vitro have been elucidated, and they include dV as part of the catalytic core and dVI which provides the first attacking nucleophile . In particular, branching requires an adenosine in a bulged context within the dVI helix and at a fixed distance from the 3' splice site, namely 7 nt for group IIA introns and 8 nt for group IIB (3,8–10). Mutations within the dV/dVI region, particularly at the branchpoint, can shift the first splicing step from transesterification to hydrolysis, and in some cases to incorrect selection of the 3' splice site (11,12).
In contrast to most other group II introns examined to date, those in plant mitochondria do not always conform to the conventional group II secondary structural model . The core helices can have weak base-pairing and certain key features sometimes appear missing. This raises questions about the mechanistic and evolutionary pathways taken by such ‘degenerate’ introns. Interestingly, a plant chloroplast intron, trnV, which lacks a dVI bulged adenosine, is excised as a linear molecule which is generated by a hydrolytic pathway of splicing (13). The importance of the dVI structure for efficient splicing in plant mitochondria is also supported by mutational analysis of the wheat cox2 intron, in that disruption of base pairing resulted in loss of splicing as monitored by in organello splicing assays (14). In the present study, we have examined the excised physical forms of the wheat mitochondrial introns nad2 intron 4, nad1 intron 2 and cox2 intron 1, designated as nad2i1282, nad1i477 and cox2i373, respectively in the nomenclature of Dombrovska and Qiu . We provide evidence for multiple splicing pathways in wheat mitochondria.
MATERIALS AND METHODS
RT–PCR and cRT–PCR analysis of intron RNAs
Mitochondrial RNA was isolated from 24 h germinating wheat embryos (Triticum aestivum cv. Frederick) as previously described (16). To examine the sequences of lariat (or circularized) excised intron molecules, a synthetic oligomer mapping close to the 5' end of the intron was used as primer in cDNA synthesis with Superscript II reverse transcriptase (Invitrogen), which is able to cross 2'–5' bonds (17). Wheat mitochondrial RNA (5 μg) was denatured at 70°C for 10 min and cDNA was synthesized using 10 U/μl reverse transcriptase, 0.2 vol 5x First Strand buffer (Invitrogen), 10 mM DTT and 0.5 mM dNTPs incubated at 45°C for 2 h. Subsequently, this primer in combination with one mapping to the 3' end of the same intron was used in PCR amplification with Taq DNA polymerase (Invitrogen). In parallel, cRT–PCR experiments (to circularize any linear intron molecules which contain a 5' monophosphate) were performed (18) with wheat mitochondrial RNA (5 μg) being incubated with 0.6 U/μl RNA ligase (New England Biolabs), 0.5 U/μl RNasin (Promega), 50 μg/ml BSA (Promega) and 0.1 vol 10x RNA ligase buffer (New England Biolabs) overnight at 14°C, and then recovered by ethanol precipitation prior to PCR amplification. The resulting cRT–PCR products will also include any endogenously circularized molecules already present in the RNA population. Prior treatment of RNA by heating at 70°C for 10 min or boiling for 5 min did not affect the nature or yield of products. In some experiments, Superscript III reverse transcriptase (Invitrogen) was used, although it appeared less efficient in traversing 2'–5' bonds. In the RT–PCR experiments, no product was seen in the absence of RT or when DNA was used as template with the cRT–PCR primer pairs (because of their inverted orientation). The sole exception was an abundant low molecular weight product for nad1 intron 2, which was determined to be co-linear with DNA by sequence analysis.
RT–PCR or cRT–PCR products were subsequently gel-purified using UltraClean 15 (MoBio Laboratories Inc.) and either sequenced directly using Sequenase version 2.0 (US Biochemicals) and electrophoresed on urea-polyacrylamide sequencing gels or after being cloned into pGemT-Easy (Promega) plasmid vectors (19). Automated sequencing was performed by the Ottawa Health Research Institute DNA sequencing facility (OHRI) and the McGill University/Genome Québec Centre.
RNase H analysis of excised introns
Wheat mitochondrial RNA (5 μg) was incubated with intron-specific oligomers at 37°C for 30 min prior to treatment with 0.03 U/μl RNase H (Invitrogen), 1 U/μl RNasin (Promega), 0.27 μg/μl BSA (Promega) and 10 mM DTT at 37°C for 30 min (20). RNase H-treated samples were phenol-extracted and ethanol-precipitated prior to northern blot analysis. Hybridizations were performed with 32P-end-labelled (T4 polynucleotide kinase) oligomer probes designed based on wheat mitochondrial sequences. The synthetic oligonucleotides used for RT–PCR, cRT–PCR, RNase H and as hybridization probes are shown in Supplementary data (online).
RESULTS
Nature of dVI branchpoint region of plant mitochondrial group II introns
Most plant mitochondrial introns fall in the group IIA category, in which the bulged adenosine of dVI is located 7 nt from the 3'end of the intron (Figure 1A, boxed) and the two unpaired terminal nucleotides (5'-AY-3') are involved in long-range interactions including –' with the dII/III linker . Figure 1B shows such an example in nad2 intron 4, which conforms to this conventional structure and its sequence is highly conserved among plants (Figure 1B, inset box). In contrast, a subset of plant mitochondrial introns, of both cis- and trans-splicing types, deviate from the classical group II structure to varying extents (Figure 1C–H). The dVI helices of some have a lower predicted thermodynamic stability than for classical group II introns, and this can be seen by A:G mispairs in the cox2 intron (Figure 1C), adjacent A:A and G:G mispairs in nad5 intron 2 (Figure 1D), A:A mispairs in nad4 intron 2 (Figure 1F) and to a much more pronounced extent in nad1 intron 1 . It is notable that plant mitochondrial intron folding can be improved by RNA editing, which converts A–C mis-pairs to A–U pairs, although not all such mispairs in dV/dVI regions are seen to undergo editing in the introns of mitochondria (23) or chloroplasts (13), and very few of the dVI mispairs shown in Figure 1 fall in that category (Figure 1D and H, but cf. underlined lower case U in dVI of nad5 intron 2). Even those introns possessing strongly paired dVI helices, in several cases deviate from the conventional group II format of having a purine stretch opposite a string of downstream pyrimidines (Figure 1D–E, G–H). The cox2 intron contains an A:G mispair immediately distal to the dVI branchpoint, as do a number of barley chloroplast introns, yet all of those were found to be excised as conventional lariats (13).
Figure 1 Secondary structure models of domains V and VI of wheat mitochondrial group II introns. (A) Classical group IIA intron (boxed), (B) nad2 intron 4 (Y14434), (C) cox2 intron (X01108), (D) trans-splicing nad5 intron 2 (M74158), (E) nad2 intron 1 (Y14433), (F) nad4 intron 2 (X57164), (G) nad1 intron 2 (X57967) and (H) trans-splicing nad1 intron 1 (X57967). The introns shown in (B–H) are designated as nad2i1282, cox2i373, nad5i1455, nad2i156, nad4i976, nad1i477 and nad1i394, respectively in the nomenclature of Dombrovska and Qiu . Expected position of dVI bulged adenosine is indicated by an asterisk. Non-conserved positions are shaded and boxes below each wheat intron (AP008982) show homologous regions from the sequenced mitochondrial genomes of rice (Oryza sativa BA000029), maize (Zea mays AY506529), Arabidopsis thaliana (NC_001284), Brassica napus (AP006444), tobacco (Nicotiana tabacum NC_006581), and sugar beet (Beta vulgaris NC_002511), and selected introns from Oenothera berteriana (M81725, M63033) and Pisum sativum (22). Dots represent identical nucleotides and lower case underlined nucleotides show C-to-U editing and differ from Y14434 and X57164, respectively.
With respect to the lariat-generating branchpoint, the majority of plant mitochondrial introns possess a bulged adenosine at the expected position, however there are several interesting exceptions (Figure 1E–H). For example, a bulged nucleotide is completely absent from the dVI helix of nad1 intron 2 and its short tight structure is conserved among all flowering plants examined to date. In contrast, in the very weakly structured dVI of nad1 intron 1, the first candidate adenosine is located at least 11 nt from the 3' end (Figure 1H) and considerable variation in sequence is seen among plants. In the case of nad2 intron 1 and nad4 intron 2 (Figure 1E–F), although a bulged nucleotide is present at the expected location (albeit flanked by pairs of varying strength among plants) it is not adenosine (cf. nad4 intron 2) or adenosine in only some plants (cf. nad2 intron 1).
Indeed the extent of sequence variation among orthologous introns from various plants in the vicinity of the branchpoint and its impact on potential base pairing (Figure 1E, F and H, shaded residues and insets) is surprising given the crucial role of dVI in lariat formation, as well as the low rate of nucleotide substitution in plant mitochondrial DNA (24). This is not the behaviour expected of sequences under strong functional constraint. In this regard, we had previously noted that dV helices also tend to show weaker pairing than classical group II introns (23). In some cases, dV structure is improved by editing, but in other instances the mispairs either remain unedited or they are not editing candidates (cf. Figure 1D and F, boxed Cs and cox2 intron U:U). Incidentally, nad4 intron 2 and nad1 intron 1 also have aberrantly long dV loops, and this is correlated with weak dVI helices and high sequence variation among plants (Figure1F and H). Based on such examination of dVI features, we selected three cis-splicing introns from wheat for this study, namely, nad2 intron 4 which has a conventional dVI (Figure 1B), nad1 intron 2, which has a short tight dVI helix (Figure 1G), and the cox2 intron, which has an A:G mispair adjacent to the branchpoint and mispairing in dV (Figure 1C).
Atypical circularized and linear introns detected by RT–PCR and cRT–PCR
To assess the presence of lariat (and/or circularized) excised introns in wheat embryo RNA populations in vivo, we used an RT–PCR strategy to amplify and then subsequently sequence the junction regions of nad2 intron 4, nad1 intron 2 and the cox2 intron. These experiments were accompanied in parallel with RNA ligase-treated samples (cRT–PCR) to determine whether linear intron molecules might also be present in the population (Figure 2). We included rps7 mRNA as a control, as it is known to be a 5' processed transcript (25) and thus can be circularized with RNA ligase. As expected, the rps7 mRNA template resulted in an RT–PCR product of 450 bp only when pre-treated with RNA ligase (Figure 2A, lane 1 versus lane 2, arrowhead).
Figure 2 RT–PCR and cRT–PCR detection of lariat (and/or circularized) versus linear wheat mitochondrial intron molecules. (A) RT–PCR across the excised intron junction using 24 h wheat mitochondrial RNA as template, either pre-treated with ligase (+L; lanes 2, 5, 7 and 9) or untreated (–L; lanes 1, 4, 6 and 8) generated products of sizes 450, 520, 630 and 400 bp (denoted by arrowheads) for rps7 mRNA, nad2 intron 4, cox2 intron and nad1 intron 2, respectively. Size markers are shown in lane 3. (B) Southern blot of gels (shown in A, lanes 4–9) using intron-specific oligomer probes for nad2 intron 4, cox2 intron and nad1 intron 2. (C) Schematic showing positions of primers (arrows 1 and 2) used for RT–PCR and cRT–PCR in panels (A) and (B). Branchpoint is shown by A. Dotted lines indicate regions of amplification. (D–F) Direct sequencing of RT–PCR products for (D) cox2 intron (+L), (E) nad1 intron 2 (–L) and (F) nad1 intron 2 (+L) using oligomers 6, 11 and 16, respectively (Supplementary data). Note that an inverted orientation of the sequencing gel is shown for (E). Arrows show positions of the 5' terminal nucleotide of the introns, stars highlight short non-encoded A-rich stretches and the discrete non-encoded insert sequence is boxed in (E).
For nad2 intron 4, nad1 intron 2 and the cox2 intron, we observed RT–PCR products for both the RNA ligase-treated and untreated templates, and their respective sizes were as expected for excised introns (Figure 2A, lanes 4–9, arrowheads). Several larger products were also observed (Figure 2A, lanes 4, 6, 7); however only nad2 intron 4 (Figure 2A, lane 4) exhibited discrete hybridization signals with intron-specific probes in Southern blot analysis (Figure 2B, lane 4). The latter may reflect amplification products which include neighbouring nad2 sequences, whereas those generated with the cox2 intron primers appear to be due to mis-priming from an unrelated genomic region. Similarly, higher molecular weight products were reported in the analysis of barley chloroplast introns (13). The apparent shift from discrete to heterogeneous high molecular weight products when RNA ligase-treated template was used with nad2 intron 4 primers (Figure 2A, lane 4 versus lane 5) may relate to contribution from circularized nad2 trans-splicing precursor molecules. In the case of nad1 intron 2, the abundant low molecular weight species (Figure 2A lanes 8, 9) were confirmed by sequence analysis to be mis-primed products containing an internal section of nad1 intron 2 that is co-linear with mitochondrial DNA (Figure 2B, lanes 8, 9), and this is consistent with the absence of Southern hybridization signal because of the intron oligomer probe maps outside that region. Comparable results with those shown in Figure 2 were reproducibly obtained with templates from different mitochondrial RNA preparations, different cDNA preparations and when alternative primers were used for cDNA synthesis.
To determine the precise nature of the excised intron junction, these RT–PCR products were cloned and sequenced. Such data are shown in Table 1. As expected, the nad2 intron 4 junction sequence was consistent with the excised intron being a lariat (with a 6 nt tail) based on six out of six RT–PCR clones and four out of four cRT–PCR clones. At the branchpoint position, we observed mis-incorporation of adenosine instead of thymidine, as has been previously reported for templates with 2'–5' bonds (13,17,22).
Table 1 Sequence analysis of RNA-ligase treated (+L) and untreated (–L) RT–PCR clones of circularized or lariat intron junction regions for wheat mitochondrial nad2 intron 4, the cox2 intron and nad1 intron 2
In contrast, for the cox2 intron only one out of five RT–PCR clones was consistent with a lariat model of splicing (Table 1). This was unexpected as this intron contains a bulged A in dVI (Figure 1B). The other four clones displayed heterogeneous circles each missing different lengths (from 10–29 nts) from the 3' terminal region of the intron. It should be noted that 50% of the clones obtained from multiple experiments had the correct insert size but sequencing revealed heterogeneous stretches of upstream and/or downstream exon sequences in addition to all or part of the intron (data not shown). This was not seen in our examination of any of the other introns. Such species might be derived from degraded RNA molecules, yet it is curious that they appear to be endogenously circularized. In this regard, evidence has been presented for a potential cryptic 5' splice site within cox2 exon 1 of petunia (26), but that appeared to be discrete rather than heterogeneous in nature.
When the cox2 intron cRT–PCR (RNA ligase-treated) products were cloned and sequenced to assess the presence of any linear intron RNA species in the population, we found that six out of eight clones had short non-encoded polyA tracts (1–5 nt in length) and four of these were full-length introns whereas the other two lacked 3' nucleotide stretches. In addition, one clone represented a full-length circularized intron species (end-to-end) and one simply lacked 3'terminal nucleotides. The latter presumably reflects contribution from the pre-existing non-RNA-ligase treated population of endogenously circularized forms. Notably in all cases, the precise 5' end of the cox2 intron was observed. The presence of a mixed population of excised cox2 intron species was corroborated by direct sequencing of the cRT–PCR products (Figure 2D). It can be seen that the sequence is homogeneous until the 5' end of the intron, but immediately past the junction there is a mixture of A-rich sequences (Figure 2D, asterisk).
The nad1 intron 2, which contains a short tight dVI helix, showed yet a different set of features in its population of excised intron molecules and none were lariats. Rather 5/9 clones showed a discrete 5'-ACAAAAC-3' insert at the junction of full-length introns, and its relative abundance is evident in the directly sequenced population of RT–PCR products (Figure 2E, boxed nts). Of the remaining four clones, one represented a full-length circularized intron, while two clones also contained 5'-ACTGT-3' which matches the first 5 nt of nad1 exon 2, and one clone lacked the 3' terminal 32 nt of the intron (Table 1). Similar results were observed for the cRT–PCR analysis, in that 3/10 clones showed precise end-to-end circularized species, and the others also contained either the non-encoded ACAAAAC block (4/10 clones) or ACTGT (3/10 clones). When the cRT–PCR products were examined directly, the sequence after the junction (Figure 2F) appears to be somewhat more heterogeneous than what was seen for the in vivo population (Figure 2E).
Linear and circular excised introns in vivo supported by RNase H analysis
Although the above strategy provides information about the physical form of excised introns, it does not directly address their relative abundance, especially since the yields of RT–PCR products may be influenced by reverse transcriptase being less efficient at extending over the 2'–5' bond of a lariat species compared with the 3'–5' bond generated by ligation of the ends of a previously linear molecule (cf. Figure 2C). To assess the relative levels of spliced lariat (and/or circularized) versus linear physical forms of introns in vivo, wheat mitochondrial RNA was annealed with intron-specific oligomers prior to incubation with RNase H and subsequent northern blot hybridization. In our analysis, we included nad7 intron 4 which we had previously shown to be predominantly lariat, but which also exhibited a minor population of fully circularized (end-to-end) intron molecules (16,27), and as expected, the vast majority appear to be lariat rather than linear (Figure 3A, black vs. open arrowhead). In the case of nad2 intron 4, when we used two annealing oligomers prior to RNase H treatment, again the shift in size was consistent with a lariat (or circularized) excised intron (Figure 3B, black arrowhead denoting 900 nt product detected by probe), when compared with the native 1.4 kb intron (Figure 3B, black dot). A minor signal was detected at the position corresponding to a linear form (400 nt, Figure 3B, open arrowhead). Also, major products of 4.8 kb derived from the linear precursor of 6 kb can be seen (Figure 3B) in addition to the precursor and intron species which were not completely digested by RNase H.
Figure 3 Determination of in vivo abundance of lariat (and/or circularized) versus linear excised introns by RNase H analysis. Wheat mitochondrial RNA (24 h) was annealed with oligomers as shown in schematics (arrows a–h) for (A) nad7 intron 4, (B) nad2 intron 4, (C) cox2 intron and (D) nad1 intron 2, prior to RNase H treatment. Positions of oligomer probes used in subsequent northern blot analysis are shown by overlined asterisks. Size markers are shown, black dots represent the positions of native excised introns, black arrowheads indicate positions expected for hybridizing products from lariat (or circularized) intron forms and white arrowheads for linear intron forms.
RNase H analysis of the cox2 intron using two intron-specific primers revealed conversion of the 1.1 kb intron (Figure 3C, black dot) to products consistent with circular (or lariat) molecules of 600 nt (Figure 3C, black arrowhead) and linear ones of 300 nt (Figure 3C, open arrowhead), as well as a shift in the 2.6 kb precursor to a prominent species of 800 nt. The relative strengths of the hybridization signals for the linear and circular forms (Figure 3C, open versus black arrowheads) suggest that both species are rather equally represented in the population, and this corroborates the results obtained with RT–PCR and cRT–PCR (Figure 2 and Table 1).
In the case of wheat nad1 intron 2, more complex northern profiles are observed because there are multiple precursors due to its co-transcription with upstream atp6 and rps13 genes, and this results in RNA species of 6, 4.6 and 3.6 kb (Figure 3D). After RNase H treatment, when only one intron-specific primer was used (oligomer g), the signal for the excised intron of 1.4 kb shifts to a 1.2 kb product consistent with the presence of a linear species (Figure 3D, black dot versus open arrowhead). This outcome was also seen when oligomer h was used, the shift being to 960 nt (Figure 3D, open arrowhead). In both cases, the linear species appear relatively abundant. Lariat (or circular) species when converted to Y-shaped structures by RNase H, would be expected to co-migrate with the native form under these electrophoretic conditions, but there is very little signal at that position (Figure 3D, black arrowhead and black dot). The high molecular weight precursors shift to the expected sizes of 2.6 and 2.3 kb. It is worth noting that the RNA ligase (cRT–PCR) experiments (cf. Figure 2A, lane 9) might be influenced by protective structures at the intron termini, such as the 3' terminal hairpin of nad1 intron 2.
DISCUSSION
Our analysis of the cox2 and nad1 excised introns in RNA isolated from germinating wheat embryos has uncovered novel physical forms which deviate from the classical lariat structure of group II introns. In contrast to introns such as nad2 intron 4 (this work), nad7 intron 3 (21,27) and nad4 intron 3 (N. Niknejad and L. Bonen unpublished data) which possess a bulged adenosine branchpoint in dVI and are excised as lariats, our observations in the case of nad1 intron 2 and the cox2 intron support the presence of both circularized and linear molecules in vivo (Figure 4). Because the cox2 intron has an apparently conventional branchpoint adenosine, it was unexpected that the lariat form was not predominant, but instead we observed heterogeneous circular excised introns which lack various lengths at the 3'terminus. Moreover, the population included full-length linear intron molecules with short, non-encoded polyA stretches.
Figure 4 Models for plant mitochondrial group II intron splicing. (A) Conventional two-step transesterification yielding intron lariat. Upstream and downstream exons are denoted by dark and light shaded boxes, respectively. (B) Hydrolytic pathway generating linear excised intron which can further undergo (i) 3' polyadenylation, (ii) 3' exonuclease activity prior to endogenous circularization of heterogeneous molecules, , (iii) addition of discrete non-encoded block (hatched box) prior to endogenous circularization or (iv) incorrect 3' splice site selection (dashed arrow) resulting in a discrete exon fragment (ACTGT for nad1 intron 2, light shaded box) being incorporated within the circular molecule. Note that the spliced exon–exon product is only shown in panel (A). (C) Hypothetical pathways of splicing via (i) external nucleophilic attack by a discrete non-encoded block (ACAAAAC, hatched box) followed by circularization of the full-length intron, (ii) an internal intronic nucleophile for first transesterification step (dashed arrows) or (iii) the hydroxyl group of the free upstream exon (dark shaded box) providing the first attacking nucleophile, followed by the terminal pyrimidine (Y) of the intron acting as the circularizing nucleophile .
Taken together, our data for the cox2 intron support a hydrolytic pathway of splicing generating linear molecules which undergo either (i) polyadenylation at the 3' terminus or (ii) endogenous circularization after 3' exonucleolytic attack (Figure 4B). Linear molecules have also previously been reported for the Streptococcus GBS intron, which possesses a conventional branchpoint adenosine (29), and for the barley chloroplast trnV intron, which in addition is polyadenylated (13); however, the wheat cox2 intron population includes endogenously circularized molecules as well. This might occur through nucleophilic attack by 2'-OH groups at other positions within dV or dVI to generate aberrant lariats with longer tails of varying lengths (Figure 4C). However, the cloned sequences correspond identically to the genomic sequence at such putative novel branchpoints, whereas typically an adenosine branchpoint is mis-read as T by reverse transcriptase (13,17,22). Alternatively, circularization might occur via an endogenous RNA ligase. It is also not excluded that the linear molecules which we observed are derived from the debranching of lariats with subsequent protection from exonuclease attack.
The nad1 intron 2 appears to be present as linear and circular molecules in vivo, but they have quite different physical features compared with the cox2 intron (Figure 4B and C). Notably, the excised introns fall into three categories: (i) precisely full-length molecules, (ii) those which in addition have a discrete non-encoded ACAAAAC block and (iii) ones which have the full-length intron plus ACTGT (which corresponds to the first 5 nt of the downstream exon). The latter could be derived from incorrect 3' splice site selection and such alternative splicing would lead to defective mRNA molecules. Because exactly the same non-encoded nucleotide blocks were consistently seen (and with or without RNA ligase treatment), these observations differ from those for the cox2 intron and do not easily conform to a model of being generated by a polyA ragged tailing mechanism (Figure 4B). The endogenous linear molecules, which comprise the majority based on RNase H analysis, appear resistant to 3' exonuclease attack (unlike the cox2 intron) and this may be related to the short tight dVI helix . In all cases, the correct 5' splice site was seen both for nad1 intron 2 as well as the heterogeneous cox2 intron molecules. The apparent absence of exonuclease attack at the 5' end of putative linear molecules might relate to protective RNA structures or close association with proteins.
Our nad1 intron 2 data suggest a combination of splicing mechanisms which exploit various internal or external nucleophiles to explain the discrete non-encoded block (Figure 4B and C, hatched box). The latter would be analogous to group I intron splicing where an external guanosine acts as the attacking nucleophile . The origin of the non-encoded ACAAAAC block is unknown. If it is added to the 3' end of the intron, it seems unlikely to be mediated by tRNA nucleotidyl transferase or an RNA-directed RNA polymerase, as was suggested for tobacco chloroplast ndhD mRNA processing (34), since there is no obvious template. Other models consistent with a precisely full-length circularized intron (which we observed at low levels for nad1 intron 2, nad7 intron 4 and the cox2 intron) include reverse splicing or a ‘spliced exon reopening’ pathway whereby the 3'-OH of the liberated 5'-exon acts as first attacking nucleophile in trans and then the hydroxyl group of the intron terminal pyrimidine attacks the 5' splice site (Figure 4C). Interestingly, in plant mitochondria, uncoupled splicing intermediates have been observed to be relatively abundant in some cases (16) and thus might contribute to such a pathway.
The apparent loss of classical group II features of mitochondrial introns in the flowering plant lineage over evolutionary time is supported by comparisons with homologues from early-diverging lineages of land plants. For example, nad1 intron 1 in Isoetes lacustris (22,36) and the cox2 intron in Acorus calamus (37) have conventional dVI structures. Moreover, the dynamic nature of plant mitochondrial genomes can have impact on intron sequences, such as the genomic rearrangements which have led to trans-splicing. It also appears that gene conversion (or swapping) events can occur between group II core structures. For example in Figure 1, it can be seen that dV and part of dVI of nad1 intron 2 and nad2 intron 1 are identical. This also underscores the plasticity and tolerance that the splicing machinery must have in light of such deviation from conventional structure.
The mixture of physical forms of excised introns observed in this study suggests that multiple biochemical mechanisms of splicing occur in plant mitochondria, such as a hydrolytic pathway as well as unusual circularization events. Their relative contributions appear to vary among introns and it will be interesting to learn if there are developmental effects, perhaps due to differences in the availability of nuclear-encoded machinery especially early in germination when seeds leave dormancy (16). In this regard, it is interesting that the steady state levels of excised introns show marked differences during germination, typically being present at higher levels in embryos than in seedlings. The apparently ‘degenerate’ nature of the folding of these group II introns raises the possibility that additional proteins and/or small RNAs have been recruited for the splicing process in plant mitochondria.
SUPPLEMENTARY DATA
Supplementary data are available at NAR Online.
ACKNOWLEDGEMENTS
The authors thank C. Carrillo, N. Niknejad, S. Subramanian and J. Crosthwait for their earlier work on wheat and rice mitochondrial introns in our laboratory. The authors also thank Dr R. Pandeya (Agriculture and Agri-Food Canada, Ottawa) for kindly providing the wheat seeds used in this study. Financial support in the form of a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (L.B.) and an Ontario Graduate Scholarship (J.L.P.T.) is gratefully acknowledged. Funding to pay the Open Access publication charges for this article was provided by the Natural Sciences and Engineering Research Council of Canada.
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