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An Organellar Maturase Associates with Multiple Group II Introns

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An Organellar Maturase Associates with Multiple Group II Introns An organellar maturase associates with multiple group II introns Reimo Zoschkea, Masayuki Nakamurab, Karsten Lierea, Masahiro Sugiurab, Thomas Börnera, and Christian Schmitz-Linnewebera,1 aInstitute of Biology, Humboldt University,10115 Berlin, Germany; and bGraduate School of Natural Sciences, Nagoya City University, Nagoya 467-8501, Japan Edited by Alan M. Lambowitz, The University of Texas at Austin, Austin, TX, and approved December 18, 2009 (received for review August 19, 2009) Bacterial group II introns encode maturase proteins required for gesting that a chloroplast reading frame is required for splicing splicing. In organelles of photosynthetic land plants, most of the these multiple introns (7, 15, 16). As there are no other candi- group II introns have lost the reading frames for maturases. Here, dates for RNA processing factors in the well-described and small we show that the plastidial maturase MatK not only interacts with chloroplast genome, it was suggested that matK is responsible for its encoding intron within trnK-UUU, but also with six additional splicing these introns. If true, MatK would be an example of a group II introns, all belonging to intron subclass IIA. Mapping anal- group II intron maturase that has left the strict association with yses of RNA binding sites revealed MatK to recognize multiple just a single target intron, joined by the group I intron maturase regions within the trnK intron. Organellar group II introns are BI4 from yeast mitochondria, which supports splicing of two considered to be the ancestors of nuclear spliceosomal introns. group I introns (17). That MatK associates with multiple intron ligands makes it an Attempts to generate knock-out alleles of matK to more attractive model for an early trans-acting nuclear splicing activity. directly assess a function of MatK in splicing failed: no homo- plastomic mutant tissue could be obtained, a clear sign for matK chloroplast | RNA-binding protein | splicing | RNA-processing | plant being essential for cell survival (18). We therefore decided to investigate on a genomewide scale, with which RNAs MatK is roup II introns are highly structured ribozymes, found in bac- associated in vivo and whether specific target sites can be iden- Gteria and organellar genomes of plants, fungi, protists, and tified. We found that MatK associates with seven intron- some animals. They catalyze their own excision from precursor containing transcripts and provides evidence that the association RNAs (1). In addition, bacterial group II introns are mobile genetic takes place in a manner different from canonical bacterial elements. These features, as well as structural similarities with maturase proteins. nuclear introns, have led to the proposition that ancestors of modern group II introns have crossed kingdoms from prokaryotes to Results and Discussion eukaryotes via eubacterial endosymbionts, and eventually evolved MatK Is a Soluble Stroma Protein Expressed Predominantly in Young into nuclear introns (2). Whereas nuclear introns are removed by a Leaf Tissue. Taking advantage of homologous recombination in large ribonucleoprotein complex called the spliceosome, each bac- tobacco chloroplast (3), we appended an extension encoding the terial intron encodes a distinct maturase protein facilitating its hemagglutinin (HA) antigen to the 5′-or3′-end of the matK gene, splicing. The evolutionary transition from the highly specific, respectively (Fig. 1 A and B). Control transformants without maturase-driven splicing toward the complex transacting spliceo- extensions but with selection markers were also generated. Correct somal machinery is unclear. Without any “living fossils” with a integration and homoplastomy of the transgenes were verified by protospliceosome, a key question is how the emerging intron PCR, Southern hybridization, and segregation analysis (Fig. 1C, diversification was accompanied by a splicing machinery with the Fig. S1). Phenotypes of transplastomic plants were indistinguish- ability to act on multiple targets. At least initially, the already existing able from WT under standard growth chamber conditions (Fig. S1), splicing factors encoded by the invading group II introns, i.e., which indicates that neither the N-terminal nor the C-terminal HA- modified maturases, may have fulfilled this task. tag interferes with MatK function. In present day organelles, maturase genes can be found in We verified expression of the tagged matK genes by Western fungal and plant mitochondria as well as in plant chloroplasts. blotting analysis and demonstrated that MatK is localized in the Although mitochondria are not directly amenable to transgenic soluble fraction, and not the membrane fraction, of chloroplasts studies, there are a number of plant species, foremost tobacco, (Fig. 2A). We quantified matK expression during tobacco for which directed mutagenesis of chloroplast genomes is pos- development (Fig. 2B). Intriguingly, strongest MatK accumu- sible (3). All land plant chloroplasts with the exception of some lation was observed at day 7 postimbibition in very young tissue. parasitic species in the genus Cuscuta (4, 5) contain a single In older plants, MatK is barely detectable. This expression pat- maturase gene called matK in the intron of the lysine tRNA-K tern is reminiscent of activity patterns of other factors involved in (UUU) gene. matK is expressed at least in green tissue (6–8). chloroplast gene expression-like plastid RNA polymerases (19). Sequence and structural homologies are found to the RNA It suggests that MatK is instrumental in staging early chloroplast binding motif (domain X) and the reverse transcriptase domain development, whereas minute levels of MatK suffice for later typically found in bacterial maturases (9, 10). Both domains are maintenance of gene expression. involved in splicing. By contrast, domains required for the mobility of group II introns are missing in MatK. The function of matK in chloroplasts is so far unknown, but its Author contributions: R.Z., M.S., T.B., and C.S.-L. designed research; R.Z. and M.N. per- position inside the trnK gene suggests a role for splicing the trnK formed research; R.Z., M.N., K.L., and C.S.-L. analyzed data; and C.S.-L. wrote the paper. precursor. Importantly, it has been suggested that other chlor- The authors declare no conflict of interest. oplast introns are targeted by MatK as well: firstly, several This article is a PNAS Direct Submission. trnK chloroplast genomes of parasitic plants and ferns have lost , Data deposition: The sequences of all four final transformation vectors have been depos- but have retained a free-standing matK reading frame, suggesting ited in GenBank (FN396957–FN396960). additional tasks for MatK, presumably in splicing other introns 1To whom correspondence should be addressed. E-mail: [email protected]. – (4, 5, 11 13); and secondly, loss of chloroplast translation leads This article contains supporting information online at www.pnas.org/cgi/content/full/ PLANT BIOLOGY to loss of splicing for some, but not all chloroplast introns, sug- 0909400107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0909400107 PNAS | February 16, 2010 | vol. 107 | no. 7 | 3245–3250 Downloaded by guest on September 30, 2021 4410 3675 A Ex II (UUU) rps16 (UUU) Ex I 3660 5290 M 3320 rec. border trnK 1110 M trnK Ex I Ex II 4629 M 4503 4877 rec. border 4870 M matK psbA Expected size of MunI restriction fragments: 2 aadA -> HA WT = 4629 -1110 = 3520 bp MM N+ = aadA and HA tag = 4629 - 3660 +1413 (aadA) = 2383 bp N- = aadA, no HA tag = 4629 -1110 + 1413 (aadA) = 4933 bp B Ex II 1732 (UUU) rps16 (UUU) Ex I 2131 5290 M 2570 rec. border 1310 rec. border trnK 1110 M trnK Ex I Ex II 4629 M 4870 M 1735 matK psbA Expected size of MunI restriction fragments: aadA -> WT = 4629 -1110 = 3520 bp HA C+ = aadA and HA tag = 2131-1110 + 1413 (aadA) = 2435 bp MM C- = aadA, no HA tag = 4629 -1110 + 1413 (aadA) = 4933 bp C N-terminal tag C-terminal tag 123 45WT 1234 5 6 7 8 9 10 11 WT 4.9 kb 4.9 kb plus aadA, no tag 3.5 kb 3.5 kb WT 2.4 kb plus aadA and HA 2.4 kb Fig. 1. Tagging of chloroplast matK.(A) Map of the matK genomic region and description of transformation vectors for N-terminal tagging of matK (vectors pRZN+/pRZN−). Black boxes correspond to exons. Inserts are shown as open boxes. Introns are shown as thin dotted lines connecting exons. aadA, selection marker; HA, hemagglutinin tag. Bold lines indicate probe used in Southern blot experiment. Numbers refer to positions on the plastid chromosome of wild-type (WT) Nicotiana tabacum (acc. no. NC_001879). rec. border, border of chloroplast DNA fragment inserted in the transformation vector. M, MunI restriction sites relevant for Southern analysis (see C). Note that sites MunI 4629 and MunI 4870 were eliminated in pRZN+ and pRZN− for cloning strategy reasons. (B) Same as in A for C-terminal tagging of matK (vectors pRZC+/pRZC−). (C) Test for homoplastomy of transformed tobacco lines by Southern hybridization. Total DNA was digested with MunI, separated by agarose gel electrophoresis, blotted, and hybridized to a matK gene-specific probe. (Left)5′ probe for the detection of the N- terminal sequence extension of matK.(Right)3′ probe for the detection of the C-terminal sequence extension (probe position and restriction sites are shown in A and B). Identified MunI restriction fragments correlate with calculated sizes (note that Mun1 site 4629 is present in transplastomic plants, i.e., has not been removed during recombination with vectors pRZN+ or pRZN−). On the basis of these fragment sizes, it can be inferred that line 1 is homoplastomic for both the selective marker and the HA tag (arrow). Of the lines generated with the pRZC+ construct, numbers 5, 7, and 8 are homoplastomic for tag and cassette (arrows). MatK Associates with Seven Chloroplast Introns. To identify MatK- +HA/−HA, were calculated.
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