Trans-splicing of organelle introns – a detour to continuous RNAs Stephanie Glanz and Ulrich Ku¨ck* Lehrstuhl fu ¨r Allgemeine und Molekulare Botanik, Ruhr-Universita ¨t Bochum, Bochum, Germany In eukaryotes, RNA trans-splicing is an important RNA- processing form for the end-to-end ligation of primary transcripts that are derived from separately transcribed exons. So far, three different categories of RNA trans- splicing have been found in organisms as diverse as algae to man. Here, we review one of these categories: the trans-splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. Trans-spliced exons can be pre- dicted from DNA sequences derived from a large number of sequenced organelle genomes. Further molecular genetic analysis of mutants has unravelled proteins, some of which being part of high-molecular-weight com- plexes that promote the splicing process. Based on data derived from the alga Chlamydomonas reinhardtii,a model is provided which defines the composition of an organelle spliceosome. This will have a general rele- vance for understanding the function of RNA-processing machineries in eukaryotic organelles. Keywords: chloroplasts and mitochondria; group II introns; organelle spliceosome; trans-splicing Introduction Introns were first discovered in 1977 and were subsequently identified in organisms from all three kingdoms, namely prokaryotes, archaea and eukaryotes. (1–3) On the basis of their splicing mechanisms and conserved RNA-folding patterns, introns are classified into the following categories: group I and group II introns, nuclear tRNA introns, archaeal introns and spliceosomal mRNA introns. Group I introns are widely distributed in genomes of prokaryotic and eukaryotic organisms but not in archaea, (4) while the tRNA and/or archaeal introns are found in eukaryotic nuclear tRNAs as well as in archaeal mRNAs, rRNAs and tRNAs. (5) Spliceo- somal mRNA introns were exclusively discovered in nuclear genomes of eukaryotes (see Glossary), (6) whereas group II introns are restricted to chloroplasts and mitochondria of lower eukaryotes, plants and some prokaryotes. These prokaryotes belong to cyanobacterial and proteobacterial lineages and are believed to be potential ancestors of chloroplasts and mitochondria. More recently, group II introns have been discovered outside these eukaryote organelles in the genome of the archaean Methanosarcina sp. (7) and in the nuclear genome of the bilaterian Nephtys sp. (8) During splicing, introns are removed from a precursor RNA, and the concomitant ligation of exons results in the formation of a mature transcript. This process of intramole- cular ligation involves only a single RNA molecule and is called cis-splicing. In cases, however, when more than one primary transcript is involved in an intermolecular ligation, the RNA is processed by trans-splicing. So far, three different categories of RNA trans-splicing have been found in genomes as diverse as archaeans to man: the spliced-leader (SL) trans- splicing, the alternative trans-splicing and the trans-splicing of discontinuous group II introns (Fig. 1). The term SL trans-splicing describes the spliceosomal transfer of a short RNA sequence (the SL, 15–50 nt) from the 5 0 - end of a particular non-coding RNA donor molecule (the SL RNA, 45–140 nt) to unpaired splice acceptor sites on pre- mRNA molecules. As a result, diverse mRNAs, ranging from a small proportion to 100% of the mRNA population in different organisms, acquire a common 5 0 -sequence. (9) This whole process converts a polycistronic transcript into translatable monocistronic mRNAs (Fig. 1A). The phenomenon of SL trans- splicing was first discovered in pre-mRNAs from nuclear genes of trypanosomes. In this organism, the capped 5 0 -terminal sequence of SL RNAs is a mini-exon containing an AUG start codon, which is trans-spliced onto the 5 0 -end of each mRNA. (10) Since then, SL trans-splicing has been found in diverse groups of eukaryotes including ascidians, cnidarians, dinoflagellates, euglenozoans, flatworms, nematodes and rotifers; (11–13) how- ever, it has an as yet unknown evolutionary origin. Alternative trans-splicing has recently been discovered in Drosophila and mammals. In this case, exons located on separate primary transcripts are selectively joined to produce mature mRNAs encoding proteins with distinct structures and functions. Alternative trans-splicing can essentially be differentiated into intragenic and intergenic trans-splicing processes. Intragenic trans-splicing is known to occur in rat and involves exon repetitions, whereas intergenic trans- splicing was found in man and mouse and involves trans- splicing of two RNA molecules originating from two different genes (Fig. 1B). (14–16) DOI 10.1002/bies.200900036 Review article *Correspondence to: U. Ku ¨ck, Lehrstuhl fu ¨r Allgemeine und Molekulare Botanik, Fakulta ¨t fu ¨r Biologie und Biotechnologie, Ruhr-Universita ¨t Bochum, 44780 Bochum, Germany. E-mail: [email protected]BioEssays 31:921–934, ß 2009 Wiley Periodicals, Inc. 921
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DOI 10.1002/bies.200900036 Review article
Trans-splicing of organelle introns –a detour to continuous RNAsStephanie Glanz and Ulrich Kuck*
¨
Lehrstuhl fur Allgemeine und Molekulare Botanik, Ruhr-Universitat Bochum, Bochum, Germany
In eukaryotes, RNA trans-splicing is an important RNA-processing form for the end-to-end ligation of primarytranscripts that are derived from separately transcribedexons. So far, three different categories of RNA trans-splicing have been found in organisms as diverse asalgae to man. Here, we review one of these categories:the trans-splicing of discontinuous group II introns,which occurs in chloroplasts and mitochondria of lowereukaryotes and plants. Trans-spliced exons can be pre-dicted from DNA sequences derived from a large numberof sequenced organelle genomes. Further moleculargenetic analysis of mutants has unravelled proteins,some of which being part of high-molecular-weight com-plexes that promote the splicing process. Based on dataderived from the alga Chlamydomonas reinhardtii, amodel is provided which defines the composition of anorganelle spliceosome. This will have a general rele-vance for understanding the function of RNA-processingmachineries in eukaryotic organelles.
Keywords: chloroplasts and mitochondria; group II introns;
organelle spliceosome; trans-splicing
Introduction
Introns were first discovered in 1977 and were subsequently
identified in organisms from all three kingdoms, namely
prokaryotes, archaea and eukaryotes.(1–3) On the basis of
their splicing mechanisms and conserved RNA-folding
patterns, introns are classified into the following categories:
group I and group II introns, nuclear tRNA introns, archaeal
introns and spliceosomal mRNA introns. Group I introns are
widely distributed in genomes of prokaryotic and eukaryotic
organisms but not in archaea,(4) while the tRNA and/or
archaeal introns are found in eukaryotic nuclear tRNAs as
well as in archaeal mRNAs, rRNAs and tRNAs.(5) Spliceo-
somal mRNA introns were exclusively discovered in nuclear
genomes of eukaryotes (see Glossary),(6) whereas group II
introns are restricted to chloroplasts and mitochondria of
lower eukaryotes, plants and some prokaryotes. These
prokaryotes belong to cyanobacterial and proteobacterial
*Correspondence to: U. Kuck, Lehrstuhl fur Allgemeine und Molekulare
Botanik, Fakultat fur Biologie und Biotechnologie, Ruhr-Universitat Bochum,
Marchantia polymorpha, Nicotiana tabacum(23,24,26,98,99)rps12-i2 cis IIA, bi
rps12-i1 trans IIA, bi, D3 Cuscuta europaea, Staurastrum punctulatum,
Zygnema circumcarinatum(100,101)
This list contains examples that have thoroughly been analysed by cDNA and/or Northern or sequence analyses. A complete list of chloroplast
introns shows that in Chlorophyta, 6 out of 27 genomes encode nine trans-spliced RNAs. Similarly, in charyophytes such as Chara vulgaris, 4 out
of 6 genomes encode rps12 RNAs that are predicted to be trans-spliced. The same is true for the 83 embryophytes whose plastomes are
completely sequenced. An exception seem to be the ndhA and ndhH transcripts that are most probably trans-spliced in wheat (Table S1).(97)
Abbreviations and gene products: bi, bipartite intron; D1-4, domains D1-D4 of a typical group II intron; n.d., not determined; pbsA, heme
oxygenase; petD, subunit IV of cytochrome-b6/f-complex; psaA, P700 chloropyll a-apoprotein of photosystem I reaction centre; psaC, subunit VII
of photosystem I; rbcL, large subunit of RubisCO; rps12, 30S ribosomal protein S12; tri, tripartite intronaThe intron nomenclature is based on the flowering plant mitochondrial literature used by Bonen.(43)
bPrediction of the secondary structure and the classification into subclasses IIA and IIB rely on sequence analyses, based on models of Michel(6)
and Michel and Ferat.(42)
Review article S. Glanz and U. Kuck
formation of domains D2 and D3 and partial domains D1 and
D4, all of which are indicative for group II introns.(37) Plastome
sequencing of the green alga S. obliquus revealed that the
psaA gene is split into two exons, which are likewise ligated
by a trans-splicing process (Fig. 3B). This discontinuous
group II intron is located and interrupted within domain D4 at
the same position as the second trans-spliced group II intron
in the psaA gene of C. reinhardtii.(31)
The large number of so far characterised trans-spliced
RNAs allows a comparison of the sites of fragmentation within
the conserved group II intron structure. With the exception of
domains D5 and D6 (Fig. 2), of which domain D5 shows the
most conserved sequence similarity within all group II introns,
all other domains can be fragmented as listed in Table 1. As
described in the next section, this list of multipartite
chloroplast genes can be extended by a number of
mitochondrial genes showing similar fragmented group II
intron structures (Table 2).
Mitochondrial trans-splicing
Mitochondrial genomes (chondriomes) of eukaryotes show a
great variation in size, ranging from about 15 kb in Metazoans
and a few algae to about 2 000 kb in species of the
Cucurbitaceae.(38) Chondriomes that are larger than 200 kb
924
are almost exclusively found in vascular plants with the
exception of the protist species ichthyosporean Amoebidium
parasiticum with a chondriome size of >200 kb.(39) The size
difference of plant chondriomes compared to other eukaryotic
chondriomes is mostly due to the presence of an additional set
of genes, promiscuous DNAs of nuclear or plastid origin,
repetitive DNAs, and numerous group I or group II
introns.(40,41)
Our analysis of 59 sequenced algal and plant chondriomes
identified 19 genomes with genes whose pre-mRNA is
predicted to be trans-spliced (Table S2). Soon after the
discovery of several trans-spliced group II introns in
chloroplasts, mainly DNA sequencing work led to the
detection of split group II introns in a range of mitochondria.
Phylogenetic analyses demonstrated that group II introns of
plant chondriomes can be distinguished from those found in
the chloroplast genome.(17,42) In addition, sequence analyses
revealed that many mitochondrial group II introns of flowering
plants, as compared with bacterial and chloroplast introns,
show variations in the sequence, structure and/or length of
typical group II introns.(43)
Most group II introns in higher plant mitochondria are
processed by cis-splicing; however, a distinct set of tran-
scripts, encoding subunits of the NADH dehydrogenase
complex, are trans-spliced. PCR and phylogenetic analyses
of cis-homologue introns in early branching land plants such
BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.
Table 2. Distribution of discontinuous mitochondrial introns from selected organisms.
Gene Intron
Splicing
type
Intron typea,
fragmented
domain Organismb
cox1 cox1-i1 trans – Diplonema papilatum, Emiliana huxleyi(54,102)
This list contains examples that have thoroughly been analysed by cDNA and/or Northern or sequence analyses. A complete list of all organelle
introns predicted to be trans-spliced is given in the supplemental material (Table S2).
Abbreviations and gene products: bi, bipartite intron; cox1, cox3, subunits of cytochrome c oxidase; D4, domain D4 of a group II intron; nad1,
nad2, nad3, nad5, subunits of NADH dehydrogenase complex; n.d., not determined; tri, tripartite intron.aThe prediction of the secondary structure as well as the classification into the subclasses IIA or IIB are based on sequence analyses, according
to the models of Michel(6) and Michel and Ferat.(42)
bAccession numbers are given in the supplemental material (Table S2).
S. Glanz and U. Kuck Review article
as ferns, horsetails, hornworts and mosses have suggested
that trans-spliced introns might have evolved from originally
cis-arranged continuous exon–intron structures by disruption
due to DNA rearrangements.(44) These genes include
nad1,(45–47) nad2(48) and nad5(49) (see Table 2 and Fig. 3C)
and recent sequencing of the first mitochondrial genome of a
gymnosperm, the cycad Cycas taitungensis, revealed trans-
spliced group II introns within the homologous genes.(50)
Similar to their chloroplast counterparts, these mosaic genes
contain introns encoding cis- or trans-spliced primary
transcripts that are flanked by sequences showing features
of group II introns (Fig. 3C).(6,34)
The genomic organisation, e.g. the exon/intron boundaries
as well as the high degree of sequence identity, is conserved
in different organisms. For instance, the intron nad2-i2 is split
at the same position in angiosperms and shows 98%
sequence identity in exons of Arabidopsis, Brassica,
Oenothera and Triticum.(51)
Another conserved example is the third intron of the nad5
gene, which is trans-spliced in all angiosperms investigated.
BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.
However, this intron can have a bipartite or a tripartite
organisation. In O. berteriana, sequence analyses of the
tripartite organisation showed that an intronic region down-
stream of exon 3 is missing, which is encoded by a distant
genomic region named tix locus (trans-splicing intron
fragment (Fig. 3C)).(52) This tripartite structure is reminiscent
of intron 1 of the chloroplast psaA RNA from C. reinhardtii that
requires the tscA RNA in order to form the correct secondary
structure.(36) Finally, despite their sequence dissimilarities,
both tix and tscA show a highly conserved secondary
structure with fragmentation sites in domains D1 and D4 at
homologous sites.(52)
An unusual trans-splicing mechanism was predicted in
both the dinoflagellate Karlodinium micrum (Alveolata)
(cox3)(53) and the diplonemid Diplonema papillatum (Eugle-
nozoa) (cox1).(54) In D. papillatum, a member of diplonemids,
which are a sister group of kinetoplastids, a fragmented cox1
gene encoded on two different chromosomes was found.
Interestingly, the flanking regions do not exhibit any
characteristics of organelle or nuclear introns nor contain
925
Review article S. Glanz and U. Kuck
conserved sequences adjacent to coding regions, and
therefore, this trans-splicing mechanism can be predicted
to be different from those processes described above for
group II introns.(55) The second remarkable example is the
bipartite cox3 gene (cytochrome c oxidase subunit 3) from the
dinoflagellate K. micrum. Similar to the example mentioned
above, no evidence of flanking group II introns was found.
Instead, numerous inverted repeats in the intergenic
sequences, which might form secondary structures, led to
the assumption that they play a role in splicing. At the splice
site, five adenine nucleotides are found that seem to be
derived from the polyA-tail of the 50-upstream fragment.
Therefore, ligation of exonic sequences seems to occur
without involvement of group II intron sequences, and the
exact mechanism of the splicing process has still to be
resolved.(56)
Trans-acting factors
Although some group II introns exhibit autocatalytic splicing
activity in vitro (see Glossary), both cis- and trans-splicing
introns require cofactors for efficient splicing in vivo.(57) In
principle, factors encoded by organelle or nuclear genomes
can be distinguished, and most of our current knowledge
stems from work with mutants having a defect in RNA
splicing.(34,58) The organelle-encoded components can be
differentiated into RNA and protein factors (Table 3). As
already mentioned above, the tscA RNA from algal chlor-
oplasts and the tix RNA from plant mitochondria are the only
Table 3. Examples of nuclear-encoded factors controlling trans-splicing
Affected
RNA Gene Organism Local. Function
nad1 OTP43 A. thaliana(78) mt trans-splicing of
psaA Raa1 C. reinhardtii(80) cp, m trans-splicing of
(class B, 30 en
RNA; group II
Raa2 C. reinhardtii(92) cp, LDM trans-splicing of
(class A; grou
Raa3 C. reinhardtii(89) cp, sþm trans-splicing of
(class C; grou
Rat1 C. reinhardtii(88) cp, m trans-splicing of
(class C, 30 en
group IIB)
Rat2 C. reinhardtii(88) n.b. trans-splicing of
(class C, 30 en
group IIB)
rps12 ppr4 Z. mays(79) cp, s trans-splicing of
biogenesis of
An extended list of trans-acting factors is given in the supplemental mat
Abbreviations: cp, chloroplast; LDM, low-density membrane; local., local