Nucleic Acids Research

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Nucleic Acids Research 27:3866-3874 (1999)
© 1999 Oxford University Press

Article

Comparative analysis of splicing of the complete set of chloroplast group II introns in three higher plant mutants

Jörg Vogel, Thomas Börner and Wolfgang R. Hess^a

Humboldt-Universität Berlin, Biologisches Institut/Genetik, Chausseestraße 117, D-10115 Berlin, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The barley mutant albostrians and the maize mutants crs1 and crs2 are defective in the splicing of various plastid group II introns. By analysing tRNA precursors and several mRNAs not previously examined, the investigation of in vivo splicing defects in these mutants has been completed. The albostrians mutation causes the loss of plastid ribosomes resulting second­arily in a disruption of splicing of all subgroup IIA introns in the chloroplast. Thus MatK, the only putative chloroplast intron-specific maturase of higher plants, might have evolved to function in splicing of multiple introns. We show that in the case of tRNA-Ala^UGC the first step of splicing is affected, as suggested by the absence of lariat molecules. Thus the plastid-encoded splicing factor lacking in albostrians must participate in the formation of the catalytically active structure. In contrast, a mutation in the nuclear gene crs1 prevents splicing of only one intron but causes specific additional effects as precursor transcripts for tRNA-Ile^GAU, tRNA-Ala^UGC, tRNA-Lys^UUU and tRNA-Val^UAC, but not tRNA-Gly^UCC, have significantly enhanced steady-state levels in this mutant. Our data provide evidence for a variety of splicing factors and pathways in the chloroplast, some encoded by nuclear and some by chloroplast genes, and possibly for a dual function of some of these factors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chloroplast RNA metabolism is a complex process in which multicistronic transcripts are processed by protein factors that are predominantly encoded by nuclear genes. As an essential step during plastid transcript maturation, splicing has to ensure the removal of 17–20 introns. Except for one group I intron, all intervening sequences in higher plant chloroplast RNAs are classified as group II. Most of the knowledge of this intron class has been obtained by detailed studies of two selected models, introns ai5 and sc.b/1 of yeast mitochondria (1,2). These are active in vitro, catalysing their own excision in a two-step transesterification pathway chemically similar to nuclear pre-mRNA splicing. The analysis of processing intermediates of several other group II introns of plastid, mito­chondrial and bacterial origin has confirmed that splicing proceeds via the same pathway in vivo (3–7). Group II introns from all different sources are characterised by a conserved secon­dary structure that is assumed to allow the intron RNA to fold into a catalytically active form. Genetic evidence indicates, however, that even introns that are capable of self-splicing in vitro require accessory factors to splice efficiently in vivo. In addition, some group II introns appear to lack structural features required for efficient self-splicing and therefore may depend on protein factors for their excision in vivo. Such accessory factors could act in various manners; for example, they could substitute for structural defects in the ribozyme core or simply facilitate intron folding. While some splicing factors are encoded within the intron (maturases), there are a growing number of examples of splicing factors that are encoded elsewhere and that are unrelated to one another (8,9).

The analysis of suitable mutants can be valuable for identifying trans-acting factors involved in the maturation of RNA in vivo. Two mutations in nuclear genes of maize have been described that affect the splicing of subsets of group II introns in the chloro­plast. Of 14 introns studied, the maize crs1 gene is required only for the splicing of atpF. In contrast, the maize crs2 gene is required for splicing of most subgroup IIB but no subgroup IIA introns (10). In order to explore whether chloroplast gene products are involved in splicing of chloroplast introns, mutants lacking plastid ribosomes (barley albostrians, Saskatoon, albina e¹⁶ and d¹³, maize iojap and chloroplast modifier) have been used (10,11). Specific splicing defects were observed in these mutants: all of the investigated group IIA, but none of the investigated IIB, introns remained unspliced (10–12).

In this study we report the completion of the aforementioned work by analysing all group II introns that had not previously been investigated in crs1, crs2 and albostrians. While prior work with these mutants focused on pre-mRNA splicing, the majority of introns examined here reside in tRNA genes. With only one exception (13), the occurrence of group II introns in tRNAs appears to be limited to plant chloroplast genomes. Our results indicate that at least two protein factors are specifically involved in plastid tRNA splicing.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plant material
The albostrians mutant of barley (Hordeum vulgare L.) is characterised by a nuclear induced plastid ribosome deficiency (14). The offspring consists of ~80% green-white striped, 10% pure white and 10% normal green seedlings. Only white tissue contains ribosome-deficient plastids. Seedlings were raised and harvested as previously described (11). The material was carefully checked to avoid cross-contamination with small areas of white or green tissue. Single plants used for cDNA synthesis were of one tissue type only.

The maize mutants crs1m1 (here crs1), crs2m1, crs2m2 and crs2m3 have been obtained by transposon tagging and were described previously (10). Material (RNA) from the characterised mutant seedlings was provided by Alice Barkan and co-workers. Crs2m2 and crs2m3 were previously referred to as crs* (10) and are weaker alleles than crs2m1.

DNA isolation, PCR and sequencing
Total plant DNA was extracted according to published protocols (15). PCRs were performed using 5 ng DNA, 10 pmol of each primer, 250 µM each deoxynucleotide triphosphate, 2 U Taq DNA polymerase (Perkin Elmer, Fullerton, CA) and 1x Taq buffer containing 2.5 mM MgCl₂. Following denaturation at 94°C for 2 min, the reaction mixtures were heated to 94°C for 1 min, 55–60°C for 1 min and 72°C for 1 min. After incubation for 30 cycles the final step at 72°C was extended for 5 min. PCR fragments were cloned into a pUC57 vector (MBI Fermentas, Vilnius, Lithuania) or were sequenced directly. PCR products for direct sequencing were purified from primers and nucleo­tides by extraction via a Qiaex spin column (Qiagen, Hilden, Germany). Sequencing was carried out using a thermal cycle amplification system (Bio-Rad, Hercules, CA) and analysed on an ABI 373 automatic DNA sequencer (Perkin Elmer).

Cloning strategy, expression of recombinant MatK and production of antisera
The full-length reading frame encoding MatK was PCR amplified from barley cDNA using the oligonucleotides MATK7xSO1 and MATK7xAS1 (see below). After cloning in PCRII (Invitrogen), the insert was released as a 1.6 kb NdeI fragment and recloned in expression vector pET15B (Novagen) as an N-terminal translational fusion with a 6xHis tag. To obtain the N-terminal, 274 residue region, the central and 3" part of matK was removed as a 0.8 kb BamHI fragment. Following gel elution and self-ligation, transformants were obtained in Escherichia coli BL21DE3. The recombinant protein was expressed after induction by IPTG for 3 h and purified from inclusion bodies by nickel-agarose affinity chromatography under denaturing conditions (6 M guanidine-HCl). Polyclonal antisera to the protein were raised in two rabbits each by a commercial producer (Biogenes, Berlin, Germany). A basic immunisation with 100 µg of the purified recombinant protein was followed by two boosts with new protein.

Isolation and analysis of RNA
Total leaf RNA was isolated using the TRIzol reagent (Bethesda Research Laboratories) as described in the protocol provided by the manufacturer. Separation and analysis of RNA molecules by RNA gel blot hybridisation and RNase protection assays were performed as described previously (6).

For reverse transcription, 5 µg of total RNA was reverse transcribed with Superscript II reverse transcriptase according to the manufacturer’s instructions (Gibco BRL, Paisley, UK) using hexamer random primers. Products of reverse transcription were amplified by PCR following standard protocols described above.

In RNase protection assays and primer extension analyses, either 10 µg of total RNA from white tissue or 15 µg of total RNA from green tissue was used. Templates for the synthesis of riboprobes, including the bacteriophage T7 promoter and cDNA from green tissues, were generated by PCR using antisense primers. Single-stranded, labelled antisense RNAs were transcribed using the MAXIscript kit (Ambion, Austin, TX). Full-length RNA probes were purified by separation on denaturing polyacrylamide gels. Using an RNase protection kit (Boehringer Mannheim, Mannheim, Germany) and following the protocol supplied, total RNA was hybridised to 3 x 10⁵ c.p.m. of labelled antisense probe and subsequently treated with a combination of RNase A and/or RNase T1. Reactions containing 5 µg of yeast tRNA served as negative controls. Loading comparable amounts of radioactivity per lane, protected fragments were separated on polyacrylamide–urea gels and visualised by exposure of the gel to X-ray film.

Oligonucleotide primers and probes for RNA analysis
Oligonucleotide sequences were designed based on existing data from the genomic region in barley chloroplasts containing trnK (16,17) or on sequence data obtained during the course of this study: trnI, trnA, trnV, trnG and atpF. All primers were obtained from Eurogentec (Seraing, Belgium).

Oligonucleotides used during construction of expression plasmids were: MATK7xSO1, 5"-GAGATATACCTAGG­G­C­C­ATATGGAAAAATTCGAAGGGTATTCAG-3"; MATK7xAS1, 5"-GCGGATCCTCGAGTTACATATGATT­A­AGAGG­GTTG­A­C­CAGGTCATTG-3".

Oligonucleotides used for RNA analysis were: ATPFEX1S, 5"-GCTAATAAATCTAACTGTAGTGGTTGGTG-3"; ATPFT7AS, 5"-(T7)-GCACCCTCTTCTTATTTATCCG-3"; ATPFINS3, 5"-GT-AAGATAGAGGATAGGCTCATTACTTATAC-3"; ATPFANT2, 5"-GACTTTTCTAATTGTTCC­A­A­A­CTAATAGAAGT-3"; PETBINS3, 5"-GCTATCTTCAAGG­C­AAATCGACC-3"; PETBANT2, 5"-GCCTCGGTCATTATGTATTGAACC-3"; TRNAT7AS, 5"-(T7)-CTTGTACCTG­A­A­CCGGTGGCTCAC-3"; TRNA-W3, 5"-CATGCATACTCC­A­CTTGGCTCGG-3"; TRNA3E1A, 5"-TGGAGATAAGCGG­A­CTCGAACCGC-3"; TRNA5E1S, 5"-GGGGATATAGCTC­A­GTTGGTAGAGCTCCG-3"; TRNAIAS1, 5"-GGTACAAG­A­TACTATCATTACCGCCTGG-3"; TRNAINS2, 5"-GTTG­C­T­CTTTGGAGAGCACAGTACG-3"; TRNGE1S1, 5"-GCGG­G­TATAGTTTAGTGGTA-3"; TRNG3TAS, 5"-GTTTAAC­A­T­GTTTAGACAAATAGAAT-ACTCC-3"; TRNI5E1S, 5"-GG­GCTATTAGCTCAGTGGTAGAGCGCG-3"; TRNI3E1S, 5"-TGGGCCATCCTGGACTTGAACC-3"; TRNVSENS, 5"-AG­GGCTATAGCTCAGTTCG-GTAGAGCAACTC-3"; TRNVANTI, 5"-GGGCTATACGGATTCGAACCGTAGACCTT-3".

Templates for in vitro transcription of single-stranded antisense probes were generated by means of PCR either including a T7 promoter core sequence in the antisense primer or using Ambion’s (Austin, TX) Lig’n Scribe Kit for subsequent ligation of T7 adaptors. Oligonucleotide pairs, PCR templates and lengths of the amplified PCR products are given in Table 1.

View this table: [in this window] [in a new window]   Table 1. Templates for in vitro transcription of single-stranded antisense probes

 
Intron-containing transcripts of trnV and trnG were specifically probed with antisense transcripts generated from templates that were obtained during lariat RT–PCR experiments (6). For trnA and trnI intron probes, a 1.8 kb PCR product spanning both genes and the intergenic spacer was cloned into vector PCRII to yield plasmid pHVtrnIA. Intron-specific DNA probes of 329 bp (trnA) and 429 bp (trnI) were released as restriction fragments using BamHI + SacI and BamHI + NcoI, respectively, and radio­labelled to high specific activity by random priming using the Rediprime kit (Amersham). Riboprobe TRNKT7AS antisense to the 5" exon of trnK and 195 nt of the 5" intron has been described previously (18).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Processing and splicing of trnA, trnI, trnV, trnK and trnG precursor transcripts in crs1, albostrians and crs2 mutants: none of the group IIA introns are spliced in albostrians plastids
The coding regions for 16S and 23S rRNA in the ribosomal rRNA operon of higher plant chloroplasts are separated by a spacer that encodes for two tRNA genes. Both of these genes, trnA and trnI, are split by subgroup IIA introns. Co-transcription of the genes in the rrn operon results in large polycistronic precursors from which monocistronic molecules of each gene are subsequently released (19).

Figure 1 shows RNA gel blots of transcripts of the trnI and trnA genes. The filters were probed with either of the two intron-specific probes or with cDNA probes corresponding to each spliced tRNA. A 7.5 kb putative precursor transcript was detected by all four probes (Fig. 1). This is presumably the precursor molecule containing the ribosomal RNAs. In contrast, smaller precursors in the size range 0.8–1.0 kb appeared to be specific for either gene. We suppose that these represent individual monocistronic, intron-containing precursors. Transcripts of comparable size have, in part, been described pre-viously (19). The lower band of the doublet obtained with the trnA intron probe must represent the released intron since it was not detected with the corresponding cDNA probe. Moreover, it was missing in the sample from ribosome-deficient albostrians plastids in which it is not spliced and in crs2m1 in which splicing of this molecule is extremely reduced. The absence of this band in crs1 in which splicing of trnA occurs points to the aberrant stabilities of several different RNA molecules in this mutant (see below).

View larger version (53K): [in this window] [in a new window]   Figure 1. Splicing of trnI–trnA precursor transcripts. RNA gel blot analyses were performed with probes specific for either the respective intron or the spliced tRNA as indicated. The analysed RNA samples were: WT, maize wild-type; yt, yeast total tRNA; G, green barley albostrians; W, white barley albostrians; crs1, maize crs1 mutant. The different crs2 alleles crs2m1, crsm2 and crs2m3 have been labelled 2m1, 2m2 and 2m3, respectively. A schematic display of the trnA and trnI gene arrangement (exons black, introns light grey) is given at the top of the figure including the intron sizes in barley and maize, respectively.

 
Different sizes for tRNA-Ile^GAU precursors in maize and barley corresponded well with the different intron lengths in these species (806 versus 950 nt in barley and maize, respectively). Crs1 mutants accumulated increased levels of the 7.5 kb precursor and of the smaller trnI but not the trnA precursor (Fig. 1). The accumulation of mature tRNAs indicated that trnA and trnI introns are efficiently spliced in all plants tested except for crs2m1 and albostrians mutants. In crs2m1 this is likely to be an indirect effect due to its severe ribosome deficiency, as can be concluded from the comparison to the weaker crs2m2 and crs2m3 alleles (10).

Ribonuclease protection assays shown in Figure 2 confirmed the observed block of splicing in albostrians plastids. Using a probe antisense to the 5" region of trnA, unspliced precursor molecules (fragments of 142 and 119 nt length) as well as 5" exon RNA without an intron (38 nt fragment) were detected with RNA from green barley chloroplasts (Fig. 2A). The protected fragment of 142 nt in wild-type RNA represents 5"-extended, intron-containing molecules that have not yet undergone cleavage at the 5"-end of the tRNA moiety by RNase P. In white albostrians plastids, however, the amount of this transcript species is extremely reduced, presumably because the total amount of precursor RNAs to trnA is significantly lower in white albostrians plastids (see Fig. 1 for comparison) whereas the activity of RNase P is comparable to wild-type plastids. In contrast to RNA from wild-type chloroplasts, no spliced molecules were detected in albostrians white plastids. Additionally, lariat protection assays (6) allowed us to check for the possible presence of trnA lariat intermediates in albostrians (Fig. 2B). Lariats are stable molecules that could accumulate even if spliced products were quickly degraded (3). Bands corresponding to the protected 5" or 3" part of the intron RNA were obtained with RNA from both green and white albostrians plastids. In contrast, an 184 nt protected fragment that could only occur if the intron 5"-end is ligated to the 2"-OH of an internal adenosine residue (‘bulging A’) did not appear with the RNA from white albostrians mutant plastids (Fig. 2B). Thus, removal of this tRNA intron is entirely inhibited at the first step of splicing in the absence of plastid ribosomes.

View larger version (42K): [in this window] [in a new window]   Figure 2. (A) Splicing of barley trnA as assayed by RNase protection. The location of the antisense probe is schematically indicated by an arrow. G and W, RNA from green and white barley albostrians seedlings, respectively; tRNA, yeast total tRNA. Sizes of M1 and M2, two different sets of marker molecules, are in nt. (B) Lariat protection of trnA. Lengths of selected molecular weight markers are given in nt. P, probe; G, W and yt as in Figure 1. Expected size and localisation of the protected fragments representing the intron lariat (L) and the 5" (5" Int.) and 3" (3" Int.) parts of the intron are shown schematically to the right. Two different conditions were used during incubation with RNase (5 U RNase ONE at 22°C or 10 U at 30°C).

 
Analysis of precursors for tRNA-Val^UAC is intriguing (Fig. 3). The unspliced precursor of 650 nt was easily detectable in wild-type barley and maize as well as in the crs1, crs2m2 and crs2m3 mutants. Similar to the results obtained for trnI, accumulation of this precursor was increased in crs1 mutants. In contrast, no or only very low levels of transcripts were detectable in albostrians and crs2m1, respectively (Fig. 3). Chloroplast genes are transcribed by a plastid-encoded RNA polymerase and one or more nuclear-encoded RNA poly­merases (20). Using the ‘–10/–35’ type promoter element found upstream of its 5"-end (21,22), the trnV gene may be exclusively transcribed by the plastid-encoded polymerase, which cannot be synthesised if plastid ribosomes are lacking. Hence, very low transcript levels prevented us from making safe conclusions about the splicing of tRNA-Val^UAC in these two mutants.

View larger version (54K): [in this window] [in a new window]   Figure 3. RNA gel blot analysis of tRNA-ValUAC. (Left) Hybridisation with a 124 nt single-stranded RNA probe recognising intron sequence. (Right) Hybridisation with a cDNA-derived single-stranded RNA probe. The positions of precursor (P) and mature tRNAs (tRNA) are shown. The sizes of marker molecules are given in kb; other lanes are labelled as in panel 1.

 
Previously it was demonstrated that the trnK intron is not spliced in albostrians white mutant plastids but is spliced at a low level in crs2m1 (10,18). Figure 4 shows that in crs1 mutant plastids the intervening sequence in the trnK gene is removed and mature tRNA is easily detectable. Interestingly, the two major precursors, 2.6 and 4.3 kb in size, accumulate to an increased level. This aberrant transcript pattern is not a consequence of the ATPase complex deficiency in the crs1 mutant (10), since a different mutant that lacks the ATPase complex (atp1-1) did not overaccumulate trnK precursors. Thus, we have provided evidence for altered processing of precursor transcripts of at least four different tRNAs: trnI, trnV, trnK and (one of two precursors) trnA in crs1. In contrast, one out of the two precursors to trnA (cf. Fig. 1) and the precursor to trnG (Fig. 5) accumulate in crs1 to the same level as in maize wild-type plastids (Fig. 5), excluding a general overaccumulation of all tRNA precursors.

View larger version (33K): [in this window] [in a new window]   Figure 4. Gel blot analysis of tRNA-LysUUU precursor processing in the maize mutants atpF1-1 and crs1 compared to wild-type barley and maize. (A) Location of the trnK gene, the intron therein and of the single-stranded RNA probe (P) used for hybridisation. (B) RNA gel blot hybridisation of trnK precursor transcripts. The probe recognises all 5" exon- and intron-containing processing intermediates as well as the mature tRNA. The length of molecular weight markers is indicated in kb.

 

View larger version (46K): [in this window] [in a new window]   Figure 5. Analysis of trnG transcripts. (A) RNA gel blot hybridisation for the presence of precursors, processing intermediates and of mature tRNA(Gly)UCC in maize mutants and barley albostrians (designations as in Fig. 1). A longer exposure of the section containing trnG splice precursors is given below the blot probed with an intron-specific antisense transcript. (B) Reconstruction experiment demonstrating the presence of spliced forms of tRNA-GlyUCC in albostrians white mutant plastids but not in crs2m1 plants.

 
The intron of tRNA-Gly^UCC is of particular interest. Its insertion site between the anticodon and the D-stem is unique among organellar tRNA introns. It is also set apart from all other group II introns in chloroplast tRNA genes in that it belongs to subgroup IIB. Comparison of trnG intron sequences from maize (23) and barley (EMBL accession no. AJ011807; this work) with a length of 679 versus 680 nt indicates that transcripts of very similar size can be expected in both organisms (Fig. 5). Precursors to tRNA-Gly^UCC of ~750 nt were detected in both barley and maize. The amount of precursor and spliced tRNA is very low but detectable in white albostrians plastids. Transcripts of trnG are efficiently spliced in crs1, whereas neither precursor RNA nor spliced tRNA-Gly^UCC was detectable in crs2m1 (Fig. 5B). Since low amounts of spliced and unspliced trnG molecules were observed in the other two crs2 alleles, we conclude that the transcription or stability of these RNA species is severely affected in this mutant.

To complete the analysis of splicing defects in albostrians, removal of the atpF intron (group IIA) and the petB intron (group IIB) from the respective precursor transcripts was examined. For this purpose, RNase protection assays involving probes that span the 3" splice junction were performed (Fig. 6). A 226 nt fragment corresponding to the spliced 3" exon of atpF is readily detected with RNA from green barley plastids (Fig. 6). In white albostrians plastids, however, the same signal is missing in spite of equal signal intensities for unspliced precursor RNA (adjusted by loading of different amounts of protected RNA). Therefore, a lack of plastid translation disrupts splicing of the atpF mRNA in barley. In contrast, the petB intron was found to be detectably spliced in both plastid types (Fig. 6). However, the level of the 220 bp fragment representing the spliced 3" exon is highly reduced in RNA from white albostrians leaves as compared to green wild-type leaves. Thus removal of the group IIB intron in petB transcripts is also affected by ribosome deficiency but is not entirely blocked, as observed for all group IIA introns investigated.

View larger version (42K): [in this window] [in a new window]   Figure 6. The products of a RNase protection experiment detecting the precursor (P) and spliced 3" exon (E) of atpF in green (G) and white (W) albostrians barley are shown adjacent to the undegraded probe (P) and a control with yeast total tRNA (tRNA). Detection of the petB precursor (P), 3" exon (E) and intervening sequence (I) by RNase protection. Sizes of marker RNAs are indicated in nt. A scheme for the probes used and the expected fragments is given at the top of the figure.

 
The matK-encoded polypeptide is lacking in albostrians plastids
One candidate for a plastid-encoded splicing factor is MatK, encoded by the ORF located within the intron of trnK (compare Fig. 4). Information on the accumulation or regulation of this putative splicing factor is scarce. Previously published data indicated that it accumulates to a rather low level, precluding direct purification (24). We therefore expressed a major part of the matK gene as a recombinant protein in E.coli and raised antibodies against it for western analysis. We chose 274 amino acids of the MatK N-terminus since this region shows a high degree of sequence variability among higher plant species. In contrast, both the RT domain and the functionally important X domain, also present in the plant mitochondrial mat-r protein (25) and in several other maturase-related proteins (26), were excluded. The serum detected the recombinant protein in control extracts from E.coli (Fig. 7). In an extract from green barley chloroplasts, a protein of an apparent molecular mass of ~60 kDa was specifically recognised. This is in agreement with the expected molecular mass of 56 kDa but different from the size of a MatK homolog of potato chloroplasts (24) and the estimated size for the corresponding polypeptide from mustard (27). No MatK was detected in white albostrians plastids. The absence of this protein correlates with the failure to splice group IIA introns in the chloroplast, consistent with its proposed role as a splicing factor for these introns.

View larger version (90K): [in this window] [in a new window]   Figure 7. Western blot analysis of barley plastid proteins for the presence of maturase K (G, green chloroplasts; W, albostrians white plastids from mutant leaves). An antiserum raised against a recombinant MatK fragment (E.coli), comprising 274 amino acids of the N-terminus, gave rise to a single band of ~60 kDa in extracts from green but not white leaves. Escherichia coli total protein extracts either containing the recombinant protein after induction by IPTG (ind.) or uninduced (contr.) were used as controls. The sizes of proteins serving as molecular weight markers are shown on the right (in kDa).

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the three best-characterised higher plant mutants with splicing defects, albostrians, crs1 and crs2, previous studies of chloroplast introns had been restricted mainly to those in mRNA precursor transcripts (10–12). Here these analyses have been continued by investigation of the remaining group II introns of which four disrupt plastid tRNA genes. We now have complete information about the different mutant phenotypes with regard to both plastid mRNA and plastid tRNA splicing.

Lack of plastid translation was shown here to affect the removal of both subgroup IIA introns and a subgroup IIB intron (petB). However, a clear non-splicing phenotype is limited to the introns in atpF, trnA and trnI, all of which belong to subgroup IIA. The same observation was made previously (10–12,18) with the structurally related introns rpl2, rps12 cis and trnK. While removal of the subgroup IIB intron in trnG was found to proceed as efficiently in the absence of ribosomes as in wild-type plastids, the level of spliced 3" exon was drastically reduced for petB transcripts in white albostrians leaves. Previously, splicing of other subgroup IIB introns, e.g. ndhA (10) or rps12 trans (12), was observed to be reduced to a considerable degree in the absence of plastid ribosomes. Therefore, it appears to be a salient feature that all IIA introns require a factor for efficient splicing that is lacking in ribosome-less plastids whereas splicing of some of the IIB introns may depend only partially on this activity.

Splicing of atpF
Analysis of all intron-containing pre-mRNAs in crs1 plants revealed a splicing defect that appeared to be confined to a single intron, i.e. atpF (10). At the same time, intron removal from atpF transcripts was found to also require a plastid-encoded factor (10; this work). We show here that splicing of the four tRNA introns not previously examined is not affected in crs1 mutants, thereby confirming that it is indeed only the splicing of atpF that is dependent upon Crs1p activity. In two previous studies, the atpF intron was targeted by a transplastomic approach: transfer of the spinach atpF intron into Chlamydomonas chloroplasts resulted in a non-splicing phenotype (28). In contrast, insertion of the tobacco atpF intron in a reporter gene and transformation into chloroplasts of the same species showed, albeit with somewhat reduced efficiency, complete and accurate intron removal from the mRNA of the reporter gene (29). Thus, atpF splicing depends on one or several host-encoded factors that must recognise the intron sequence or structure that are specific for higher plant chloroplasts. It is interesting to note that one such factor is nuclear encoded and specific for this particular intron whereas a second is plastid encoded and most likely involved in splicing of several more introns (10; this paper). One potential role for splicing factors is to compen­sate for structural defects that have accumulated in the RNA core of a self-splicing progenitor intron. We modelled the secondary structure of the barley atpF intron (not shown, available upon request) and found that the atpF intron structure does not appear to be less conserved than other chloroplast introns. Thus, dependence on multiple splicing factors is not necessarily driven by severe losses of essential intron features. Furthermore, considering the limited genetic analyses to date, this result suggests that multiple splicing factors might be required for other chloroplast introns as well.

Crs1p may act as a bifunctional protein
Frequently, factors assisting in the splicing of group II and group I introns serve an additional function in other cellular processes. Examples include the yeast MRS2 protein involved in assembly of components of the respiratory chain (8), the Neurospora mitochondrial tyrosyl-tRNA synthetase (30) and the yeast DEAD-box protein MSS116 (31). Our results suggest that Crs1p may also be a bifunctional protein. In addition to their defect in atpF splicing, crs1 mutants accumulate increased levels of precursors to several tRNAs (Figs 1, 3 and 4). Thus Crs1p may play a further role in plastid gene expression. In fungal mitochondria, several splicing factors were recruited from the pre-existing pool of RNA binding proteins. Some of them, such as the Neurospora CYT-18 protein, adopted a role in splicing on the basis of its initial function in maturation of tRNA transcripts (32). A similar situation may exist for Crs1p.

MatK as a splicing factor
The splicing factor(s) lacking in albostrians mutant plastids are likely to be encoded by a plastid gene. It could be a ribosomal protein with RNA chaperone activity, like the E.coli proteins Rps12 and StpA, which facilitate group I intron splicing in vitro (33,34). Alternatively, it could be the ribosomes themselves, as described for splicing of the group I intron of the T4 thymidylate synthase gene (35). However, the latter possibility seems unlikely given that four of the introns that are unspliced in albostrians white plastids reside in tRNAs and are not translated.

A factor previously proposed as a possible plastid-encoded splicing factor is MatK (18,24,27). This idea, although appealing, has so far received only circumstantial evidence. The matK gene is conserved among all higher plants. Furthermore, in barley and maize an evolutionarily ancient amino acid motif is restored by RNA editing (18). The plastids present in white albostrians leaves are ribosome deficient. Hence, all plastid-encoded proteins should be lacking, including MatK. We have confirmed this expectation here, by using an antibody to show that MatK is indeed absent from albostrians mutant plastids. Hence, it is tempting to assume that MatK has evolved from a trnK intron-specific maturase to function in splicing of several introns, i.e. all plastid group IIA introns of higher plants, para­lleled by the loss of several of the domains known from other maturases. This, in turn, would resemble a process thought to have taken place during the evolution of the spliceosomal apparatus (36). Nevertheless, the possible existence of other plastid-encoded proteins participating in the splicing process of group IIA introns cannot be excluded.

Specificities of known plastid splicing factors
Previously, evidence was presented that the crs2 gene functions specifically in the splicing of group IIB introns: of 14 introns examined, nearly all of the IIB introns, but none of the group IIA introns, are dependent on crs2 function (10). The results presented here complete the analysis of crs2 splicing defects by analysing the remaining tRNA precursor transcripts. They support the previous conclusion that group IIA and IIB introns in the chloroplast require different splicing factors. The differences between IIA and IIB introns are well defined (1). Group II introns are in principle mobile genetic elements. Examples of both intron types can be found in phylogenetically widely differing organisms and organelles. The insertion of only one or a few of them into the plastids of the putative progenitors of land plants (37) might have been followed soon after by a spread into other locations of plastid DNA. Consequently, the fact that chloroplast splicing factors exist that are essential and specific for either all IIA or all but one group IIB introns might be taken as evidence for a common evolutionary origin in either subgroup. At the same time, dependence on a particular splicing factor had to be preserved. Except for the MatK–trnK interaction, all of the factors that mediate splicing in chloroplasts are thought to act in trans. By this, evolution of intron splicing in higher plant chloroplasts is set apart from bacterial and several mitochondrial group II introns that have most likely co-evolved with their cis-encoded maturases (38) and is more reminiscent of the evolution of spliceosomal introns (36).

One of the chloroplast introns, ycf3 intron 2, does not depend on any of the splicing factors whose activities have so far been identified. Hence, it is possible to classify the group II introns of higher plant plastids according to their dependence on distinct splicing factors in at least four groups: (i) those that require plastid ribosomes or a plastid gene product (all IIA); (ii) those that require both Crs1p and a plastid-encoded protein (atpF); (iii) those that require Crs2p (all IIB except one); and (iv) those that splice independently of the aforementioned factors (ycf3 intron 2).

With this work, a complete characterisation of the three plant mutants with splicing deficiencies of chloroplast introns is now available. Identification of the proteins that are lacking in these mutants and of their function will allow new insights into the process of group II intron splicing in vivo. Moreover, a mutant with blocks in tRNA splicing, i.e. barley albostrians, offers an excellent system to explore how gain and persistence of group II introns is linked to other steps of tRNA maturation.

	ACKNOWLEDGEMENTS

 
We thank Dr B. Nielsen for critical reading of this manuscript, Dr A. Barkan for the opportunity to include crs mutants in our analysis and for reading of a previous version of this paper, Ms B. Jenkins for providing crs2 RNA, Mr Brad Till for providing crs1 RNA and Mr D. McCormac for providing atpF1-1 RNA. Financial support by grant HE 2544/2-2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany, to W.R.H. and T.B. is gratefully acknowledged.

	FOOTNOTES

 
^a To whom correspondence should be addressed. Tel: +49 30 2093 8144; Fax: +49 30 2093 8141; Email: wolfgang=hess{at}rz.hu-berlin.de ⁺AF072710, AJ010570–AJ010573, AJ010977, AJ011807

DDBJ/EMBL/GenBank accession nos⁺.

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Received May 24, 1999. Revised and Accepted August 13, 1999.

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