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,

44780 Bochum, Germany.

E-mail: ulrich.kueck@ruhr-uni-bochum.de

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

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 50-

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 50-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 50-terminal

sequence of SL RNAs is a mini-exon containing an AUG start

codon, which is trans-spliced onto the 50-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)

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Figure 1. Three categories of trans-splicing. A: Spliced leader (SL) trans-splicing. SL trans-splicing accurately joins sequences derived from

separately transcribed small non-coding RNAs and independently transcribed pre-mRNAs. The SL sequence is donated from the SL RNA to pre-

mRNAs to form the 50-terminal mini-exon of the mature mRNA. Outron indicates the 50-segment of a trans-spliced pre-mRNA upstream of the

trans-splice acceptor site. B: Alternative trans-splicing. Intragenic trans-splicing generates mRNAs containing tandem duplications of specific

exons (dark blue) and intergenic trans-splicing generates chimeric mRNAs (grey and light blue) between pre-mRNAs originating from two

different genes (A or B). C: Group II intron trans-splicing. Primary transcripts derived from distantly located exonic sequences are joined end to

end and ligated after assembly and splicing of the flanking group II intron sequences. Exons are shown as boxes and waved lines represent non-

translated sequences.

Review article S. Glanz and U. Kuck

Finally, trans-splicing occurs between transcripts derived

from scrambled gene fragments flanked by discontinuous

group II introns (Fig. 1C). Group II introns are characterised by

a conserved secondary structure configuration. This structure

consists of six major stem-loops, corresponding to domains

D1–D6, radiating from a central core of single-stranded RNA

segments that brings the 50- and 30-splice junctions into close

proximity. For correct folding and catalytic function, the

formation of tertiary interactions is essential. Within group II

introns, two main subclasses of secondary structures, IIA and

IIB, each consisting of two forms (IIA1, IIA2 and IIB1, IIB2),

have been found and more recently, two further classes, IIC

and IID, have been discovered in bacteria.(6,17) A model for the

secondary structure of a typical group IIB intron is shown in

Fig. 2 and excellent reviews concerning the structure and

folding of these introns have been published.(18,19)

Discontinuous group II introns are found in chloroplasts of

algae as well as in chloroplasts and mitochondria of higher

plants and will be the focus of this review. The genes involved

consist of exons that are distributed throughout the genome

and flanked by 50- and 30-regions of group II intron consensus

sequences. Due to the assembly of these regions, a functional

group II intron secondary structure is restored in trans. In

many cases, the correct assembly and splicing reaction

seems to depend on trans-acting factors, which could be RNA

922

molecules and/or proteins. Discontinuous group II introns

share a common splicing mechanism with spliceosomal

introns and are therefore considered as an evolutionary link

between cis-splicing group II introns and nuclear spliceoso-

mal introns. These nuclear spliceosomal introns depend

functionally on the trans-acting spliceosome machinery.(20,21)

Previously, it was assumed that DNA rearrangements within

group II introns result in discontinuous mosaic genes with

exons scattered over the genome.(22)

Chloroplast trans-splicing

The plastid DNA (plastome) of plants and algae codes for

100–140 genes. Most of these genes are organised in

operons and the corresponding polycistronic precursor

transcripts undergo complex posttranscriptional processes.

These processes include the stabilisation of the RNA as well

as cis- and trans-splicing processes, endonucleolytic clea-

vage, RNA editing, and terminal nucleotide additions and/or

deletions. We have analysed a total of 120 sequenced

chloroplast genomes with regard to trans-splicing processes

(Table S1). Our analysis revealed that no trans-splicing events

take place in the three members of the Apicomplexa and in a

single cercozoan species sequenced so far, but that 101

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

Figure 2. Secondary structure model of a typical group IIB intron.

Intron and exon sequences are given as thin and thick lines, respec-

tively. Arabic numerals denote the six conserved domains of group II

introns (D1–D6). The dashed domain D4 is the most variable intron

region and sometimes contains an open reading frame. A branch

point involved in group II intron splicing is circled. Arrows indicate 50 to

30 strand polarity. Potential fragmentation sites of trans-spliced introns

are mapped with arrowheads in domain D1, D2, D3 and D4. Typical

tertiary interactions between exon-binding sites (EBS) and intron-

binding sites (IBS) are indicated. For a complete set of tertiary

interactions in group II introns see Pyle et al.(95) and Fedorova et al.(18)

S. Glanz and U. Kuck Review article

trans-splicing events take place in the other 116 analysed

eukaryotic genomes. Table 1 summarises all characterised

chloroplast trans-splicing introns known to date, and Fig. 3A

shows examples for the organisation of exon sequences in

chloroplast genomes from selected organisms. The first

examples of chloroplast trans-splicing were discovered for the

group II introns from the Marchantia polymorpha and

Nicotiana tabacum rps12 gene, encoding the 30S ribosomal

protein S12, and from the C. reinhardtii psaA gene, encoding

the P700 chloropyll a-apoprotein of photosystem I reaction

centre.(23–26)

Maturation of the rps12 mRNA represents a complex

trans-splicing process, and the corresponding gene shows a

highly similar organisation in chloroplasts of charyophytes

and embryophytes. The mosaic rps12 genes, like other

discontinuous chloroplast genes, contain introns encoding

cis- and/or trans-spliced primary transcripts that are flanked

by sequences showing features of group II introns (Table S1).

In Fig. 3A, two out of four possible organisations of the rps12

genes are depicted. In all cases, splicing of exon 1 and exon 2

occurs in trans, with an intronic fragmentation site in domain

D3, while exon 2 can harbour further two exonic sequences

that can be processed on the RNA level by cis-splicing.

Alternatively, the continuous exon 2 sequence or the

discontinuous exon 2-exon 3 sequences can be located

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

either in a large single copy region or in the two inverted

repeats of chloroplasts genomes. Both the 50- and 30-rps12

gene fragments are organised in operons and are expressed

as polycistronic transcripts.(27,28) Maturation of the rps12

mRNA comprises both endonucleolytic cleavage of the

polycistronic transcripts and trans-splicing of exon 1 with

exon 2 as well as cis-splicing of exons 2 and 3.

Numerous sequencing projects have enabled the in silico

analysis of further genes with putative trans-spliced introns

that has revealed other genes than those mentioned above.

For example, these genes include psaA of the green alga

Scenedesmus obliquus, pbsA (heme oxygenase) of the red

alga Rhodella violacea, petD (subunit IV of cytochrome-b6/f-

complex) and psaC (subunit VII of photosystem I) of the green

algaeStigeoclonium helveticum and Oedogonium cardiacum,

rbcL (large subunit of ribulose 1,5-bisphosphate carboxylase/

oxygenase) of the green algae S. helveticum and Floydiella

terrestris, and ndhH (subunit of the NA(P)DH dehydrogenase

complex) of Triticum aestivum.(29–33)

The highest number of trans-spliced introns within a plastid

genome was predicted from complete sequencing of the

chloroplast DNA from the green alga S. helveticum.(29) Four

discontinuous group II introns were identified in the pre-

mRNAs, one each in the petD and psaC genes and two in the

rbcL gene. Recently, further chloroplast DNAs from the green

algae F. terrestris and O. cardiacum were reported to encode

petD, psaC or rbcL pre-mRNAs that are spliced in trans

(Table 1, Table S1 and Fig. 3B).

To date, the best-analysed trans-splicing process is the

one of the psaA mRNA in the unicellular green alga

C. reinhardtii. This alga can be regarded as the model

organism for the analysis of plastid gene expression during

photosynthesis and the communication between the nucleus

and the chloroplast.(34) Mutant strains with a defective

photosystem I finally served as base for the identification

of trans-splicing processes.(35) As early as 1987, it was

already known that the three exons of the psaA gene are

distributed on the plastome and transcribed separately from

each other(25). Two trans-splicing steps are necessary to form

the mature mRNA. For intron 1, three independently trans-

cribed RNA molecules assemble into a functional group II

intron structure by base pairings and tertiary interactions. This

tripartite group IIB intron is interrupted in domains D1 and D4;

thereby exon 1 is flanked by a portion of domain D1 and exon 2

by the entire domains D4 and D5 as well as a portion of D6.

The rest of domains D1 and D4 as well as the entire domains

D2 and D3 are delivered from a third RNA molecule, the

plastid-encoded tscA RNA, which is 450 nt in length.(36)

The tscA RNA was also detected in C. gelatinosa (376 nt)

and in C. zebra (466 nt) and exhibits sequence identity of

approximately 55% to the tscARNA ofC. reinhardtii for both of

the species. Analysis of the secondary structure of the three

tscA RNAs showed also a high degree of similarity with the

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Table 1. Distribution of discontinuous chloroplast group II introns from selected algae, higher plants and mosses.

Gene IntronaSplicing

type

Intron typeb,

fragmented

domain Organismc

pbsA pbsA-i1 trans n.d. Rhodella violacea(33)

pbsA-i2 cis? n.d.

petD petD-i1 trans IIB, bi, D1 Oedogonium cardiacum, Stigeoclonium helveticum(29,30)

psaA psaA-i1 trans IIB, tri, D1þD4 Chlamydomonas reinhardtii(25)

psaA-i2d trans IIB, bi, D4

psaA-i1d trans IIB, bi, D4 Scenedesmus obliquus(31)

psaC psaC-i1 trans IIB, bi, D1 Oedogonium cardiacum, Stigeoclonium helveticum(29,30)

rbcL rbcL-i1 trans IIB, bi, D1 Floydiella terrestris, Stigeoclonium helveticum(29,30)

rbcL-i2 trans IIA, bi, D2 Floydiella terrestris, Stigeoclonium helveticum(29,30)

rps12 rps12-i1 trans IIB, bi, D3 Epifagus virginiana, Hordeum vulgare L.,

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)

cox3 cox3-i1 trans – Karlodinium micrum(103)

nad1 nad1-i1 trans IIB, bi, D4 Arabidopsis thaliana, Brassica napus, Oenothera berteriana,

Petunia hybrida, Triticum aestivum, Vicia faba, Zea mays(45–47,104–107)nad1-i2 cis bi

nad1-i3 trans IIB, bi, D4

nad1-i4 cis bi Arabidopsis thaliana, Brassica napus, Oenothera berteriana,

Vicia faba(47,105–107)

trans IIB, bi, D4 Petunia hybrida, Triticum aestivum, Zea mays(45,46,104)

nad2 nad2-i1 cis IIA, bi Arabidopsis thaliana, Brassica napus, Oenothera berteriana,

Triticum aestivum, Zea mays (sterile line) (48,51,108,109)nad2-i2 trans IIA, bi, D4

nad2-i3 cis IIA, bi

nad2-i4 cis IIA, bi

nad3 nad3-i1 trans IIA, bi, D4 Mesostigma viride(110)

nad3-i2 trans IIA, bi, D4

nad5 nad5-i1 cis IIA, bi Arabidopsis thaliana, Brassica napus, Oenothera berteriana,

Triticum aestivum, Vicia faba, Zea mays(49,105,111,112)nad5-i2 trans IIA, bi, D4

nad5-i3 trans IIB, bi, D4 Arabidopsis thaliana, Brassica napus, Triticum aestivum, Vicia faba,

Zea mays(49,105,111,112)

trans IIB, tri, D4 Oenothera berteriana(49)

nad5-i4 cis IIA, bi Arabidopsis thaliana, Brassica napus, Oenothera berteriana,

Triticum aestivum, Vicia faba, Zea mays(49,105,111,112)

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

determined; OTP, organelle transcript processing defect; ppr, pentatricop

psaA tscA RNA; s, stroma.

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organelle-encoded RNA factors so far known to support the

splicing process in trans.(36,52) As described in the next

chapter, the tscA RNA is most probably part of an organelle

spliceosome that similar to the nuclear spliceosome contains

protein as well as RNA components.(34)

Maturases are highly conserved organelle-encoded pro-

teins, and are usually encoded in domain D4 of some of the

characterised group II introns. These enzymes catalyse the

excision of non-autocatalytic introns, e.g. the excision of the

intron from its own primary transcript, and together with the

intron RNA, they form a ribonucleoprotein (RNP) com-

plex.(59,60) Moreover, maturases have reverse transcriptase

activity, mediating the integration of their ‘mobile’ introns into

new DNA sites (see Glossary).(61) Functional maturases

encoded by bacterial introns were shown to promote splicing,

e.g. of the group II intron Ll.LtrB from Lactococcus lactis.(62)

Recently, the Ll.LtrB intron was used as a model system to

study group II intron trans-splicing in bacteria. A highly

sensitive splicing/conjugation assay was developed and it was

demonstrated that assembly and trans-splicing of a frag-

mented group II intron can efficiently take place in bacterial

cells. The authors mimicked naturally occurring fragmentation

sites, e.g. the site in domain D1 of psaA, and further showed

that the Ll.LtrB intron-encoded maturase LtrA is essential for

trans-splicing.(63,64)

Nuclear-encoded trans-acting factors are the second class

of components that are able to compensate for the loss of

autocatalytic splicing activity in organelle introns (Table S3). It

is generally accepted that mitochondria and chloroplasts are

the result of an endosymbiosis of a-proteobacteria-like and

of group II introns.

Sequence homology

nad1 intron 1 PPR protein

psaA

d processing of tscA

B)

n.d. (possible PPR protein)

psaA

p IIB)

Pseudouridine synthases

psaA

p IIB)

Pyridoxamine 50-phosphate oxidases

psaA

d processing of tscA RNA;

NADþ-binding domain of poly

(ADP-ribose) polymerases

psaA

d processing of tscA RNA;

Domain of a putative RNA-binding

protein of Synechococcus spec.

WH8102

rps12 (intron 1),

ribosomes

PPR protein

erial (Table S3).

isation of gene products; m, membrane; mt, mitochondrion; n.d., not

eptide repeat; Raa, RNA maturation of psaA; Rat, RNA maturation of

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

Figure 3. Organisation of selected chloroplast and mitochondrial genes with trans-spliced RNAs. A: Two possible genomic organisations of

rps12 genes from different sources. The bottom example is representative for higher plants with duplicated 30-exons in the inverted repeat (IR)

regions IRa and IRb and a 50-exon in the large single copy region (LSC). B: Examples of chloroplast genes from diverse algae. C: Schemes of

mitochondrial loci from the five exons of nad1 and nad5. The transcripts of nad1 and nad5 are polycistronic (data not shown).(96) The genome of

O. berteriana was not yet completely sequenced. Exons are represented by black boxes with their corresponding size in base pairs. Arrows

indicate direction of transcription. Cis-spliced introns are depicted as red boxes and trans-spliced introns are marked in yellow. Black double

slashes indicate split gene fragments, which are separately transcribed. Distances in kb were determined from a clockwise orientation of the

chloroplast genomes. Abbreviations of species are as follows: C.v., Chara vulgaris (NC_008097); N.t., Nicotiana tabacum (NC_001879); O.b.,

Oenothera berteriana (X07566, X60046, X60049, X99516); S.o., Scenedesmus obliquus (NC_008101); S.h., Stigeoclonium helveticum

(NC_008372); T.a., Triticum aestivum (NC_007579). Abbreviated gene designations are explained in the legend of Tables 1 and 2.

S. Glanz and U. Kuck Review article

cyanobacteria-like prokaryotes, respectively. This process is

accompanied by the relocation of a major part of the

prokaryotic genomes into the chromosome of the host cell.

As a consequence, the nuclear-encoded organelle proteins

have to be retargeted to their ancestral compartments.(65,66)

In Fig. 4, this situation is depicted for the chloroplast of the

unicellular green alga C. reinhardtii. RNA-processing, trans-

lation, as well as assembly of membrane or membrane-

associated complexes, is dependent on both, organelle- and

nuclear-encoded proteins. The latter are translated on

cytosolic ribosomes and will be transported through the

chloroplast membranes into the inner space of the orga-

nelle.(67–69) Table 3 summarises nuclear-encoded proteins

involved in trans-splicing of group II introns together with the

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

functionally characterised gene products. When known, we

also give the homology of the proteins to other factors as well

as their subcellular localisation.

While some of these trans-acting factors seem to be

specific for only a single intron, other factors are involved

in splicing of a set of introns. Moreover, some of these

nuclear-encoded proteins have acquired, in addition to

their catalytic role, further organelle functions during

splicing. It is therefore assumed that during evolution some

of these nuclear-encoded factors were adapted to the

binding of intron structures, thus playing a role in the splicing

process.(22)

The trans-acting factors involved in splicing of a set of

introns can be divided into three groups. The first group of

927

Figure 4. Dependence of chloroplast biogenesis on nuclear-

encoded factors. During chloroplast biogenesis, coordination of gene

expression is achieved by nuclear-encoded factors that affect RNA

processing, translation, and also assembly of complexes. Chloroplast

multisubunit complexes are thus formed by both nuclear- (arrows) and

chloroplast- (dashed arrows) encoded polypeptides.

Review article S. Glanz and U. Kuck

nuclear-encoded factors comprises enzymes involved in RNA

maturation processes, and recently, new members of this

group with homologies to mitochondrial maturases were

detected. Four genes for group II intron maturases, nMat-1a,

nMat-1b, nMat-2a and nMat-2b, were identified in the nuclear

genomes of both A. thaliana and Oryza sativa. Interestingly,

these maturase-like proteins are not intron-encoded. The

predicted mature proteins show homology to mitochondrial

counterparts and contain putative mitochondrial import

sequences. It is assumed that they were transferred during

evolution from the chondriome to the nuclear genome and

may have retained their role in splicing of mitochondrial group

II introns, as was functionally demonstrated with an

A. thaliana mutant analysis.(70,71) Nuclear-encoded RNA-

editing factors also play a role in splicing. RNA editing in plant

organelles is mediated by specific nuclear-encoded factors(72)

and is essential for the formation and stabilisation of splicing-

competent primary and secondary structures of several

mitochondrial group II introns.(48,51) Almost all protein-coding

transcripts as well as some introns and tRNAs in mitochondria

928

of higher plants are edited. The editing event comprises

mostly C-to-U substitutions.(73) For example, fusion of the

trans-splicing intron nad1-i3 from O. berteriana with

sequences from an autocatalytic splicing intron from

yeast revealed that unedited intron sequences are not able

to form a functional, splicing-competent group II intron

structure.(74)

The second group of nuclear-encoded factors exhibit

repeated motifs of 34–38 amino acids, e.g. like in penta-

tricopeptide repeat (PPR) proteins. The PPR protein family is

characterised by tandem repeats of a motif consisting of a

degenerate 35 amino acid repeat. Several PPR proteins are

encoded in the genomes of animals, fungi and trypano-

somes,(75) and most of these proteins are found in genomes

of higher plants.(76) Already characterised PPR proteins show

RNA-binding features and affect the processing or the

translation of specific RNA molecules in mitochondria and

chloroplasts.(77) Recently, the nuclear OTP43 (organelle

transcript processing defect) gene was shown to be

specifically required for trans-splicing of mitochondrial

nad1-i1 in A. thaliana.(78) Another example is PPR4 of Zea

mays, which is responsible for trans-splicing of the first intron

of the chloroplast rps12 RNA by directly binding to this

intron.(79) Finally, Raa1 (RNA maturation of psaA) from

C. reinhardtii, which is involved in the trans-splicing process of

both psaA introns, also harbours tandem repeats similar to

those found in PPR proteins.(80)

The third and final group of nuclear-encoded factors

represent proteins that cannot be assigned to any classified

function. CRS1 (chloroplast RNA splicing 1) from Z. mays, for

example, contains a new RNA-binding domain, the CRM

domain (chloroplast RNA splicing and ribosome maturation),

which is also found in archaeal and bacterial proteins, involved

in the maturation of ribosomes.(81) This CRM domain is

likewise found in CRS2, another splicing factor from maize.(82)

Moreover, the trans-acting factors in Z. mays are known to be

part of a high-molecular-weight ribonucleoprotein complex

that also contains spliced intron sequences.(79) Similar

data are available from C. reinhardtii, which in recent years

was the subject of intense mutational analyses all of which

show a defect in trans-splicing of the psaA RNA.(80,83) As

detailed below, these studies led to the notion of a complex

chloroplast spliceosome being involved in the splicing

process.

Putative chloroplast spliceosome of themodel alga C. reinhardtii

Trans-splicing of the psaA RNA from C. reinhardtii requires a

plastid-encoded tscA RNA and at least 14 nuclear-encoded

chloroplast factors.(36,84) The corresponding nuclear mutants

are grouped into three classes according to their mode of

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

Figure 5. Trans-splicing of the chloroplast psaA RNA of C. reinhardtii. The psaA gene is fragmented into three independently transcribed

exons, which are flanked by consensus sequences of group II introns (green wavy lines). To generate a mature psaA mRNA, two trans-splicing

steps are necessary. For the formation of the first group II intron, a small chloroplast-encoded RNA (tscA) is required that interacts with precursor

transcripts. The tscA RNA is co-transcribed with chlN and is the subject of various 30 end processing events. Ovals represent nuclear mutant

classes, which are affected in different steps of the trans-splicing process. Colours indicate class A, B and C mutants, respectively. The first group

II intron is labelled as denoted in Fig. 2. Arrowheads indicate the sites of fragmentation. Abbreviations: EBS2, exon binding site 2; IBS2, intron

binding site 2.

S. Glanz and U. Kuck Review article

action (Fig. 5): class A mutants fail to trans-splice exon 2 and 3

primary transcripts; class B mutants neither splice exon 1 and

2 nor exon 2 and 3 primary transcripts; and class C mutants

are not able to splice exon 1 and 2 primary transcripts.(85,86)

Lack of correct splicing in class B and C mutants can take

place at two different levels of RNA processing, i.e.

either splicing of the primary psaA transcripts or 30-end

processing of the tscA RNA is affected. Of note is that

maturation of the tscA precursor is a prerequisite for correct

splicing of exon 1 and 2. To date, five trans-acting factors were

characterised in C. reinhardtii with three belonging to class C

factors (Table 3).

Based on cofractionations and sucrose density

gradient centrifugations using protein extracts of wild-type

or splicing-deficient mutants, Rochaix and co-workers

proposed at least three protein complexes, two of which

are associated with chloroplast RNAs. Thus, the latter two

can be considered as chloroplast RNP (cpRNP) complexes

that might be part of the chloroplast spliceosome. This

concept of a spliceosome-like complex was further supported

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

by reciprocal coimmunoprecipitations, showing that either

protein of the complex can be immunoprecipitated with the

other. However, these data do not yet answer the question

whether the identified splicing factors interact with each

other.(80,87)

Figure 6 provides a current model of chloroplast

spliceosomal complexes and their action in splicing and

integrates data from several experimental approaches. The

tscA RNA participating in the formation of the secondary

group II intron structure is involved in splicing of the first group

II intron (psaA-i1).(36) At least three factors are involved in the

processing of the precursor molecule containing the tscA

RNA. As mentioned above, Raa1 is related to PPR proteins

and is a factor involved in tscA RNA maturation. It contains

two distinct domains of which the C-terminal domain is

involved in processing of the tscA RNA, and the central

domain in splicing of intron 2. The function of both domains

was deciphered when different truncated versions of theRaa1

gene were used in restoration experiments analysing two

different mutants.(80)

929

Figure 6. Model of chloroplast psaA RNA trans-splicing complexes in C. reinhardtii. The scheme integrates data from several groups of

investigators as described in the text. Depicted are proteins and protein complexes required for trans-splicing of three psaA mRNA

precursors. The two secondary structures resemble the folding of 50- and 30-intronic RNAs flanking exon 1, 2 and 3 sequences modified

after Goldschmidt-Clermont et al.(84) For details of the secondary structure from the first intron see Fig. 5. Abbreviations: chlN, subunit of the

light-independent protochlorophyllide reductase; Cpn60, chaperonine 60; cNAPL, chloroplast nucleosome assembly protein-like; pL118B and

pL137H, class B factors, and pL121G, class A factor, which are defined genetically;(87) Raa1-6, RNA maturation of psaA; Rat1-3, RNA

maturation of psaA tscA RNA; tscA, trans-splicing chloroplast. Abbreviations are as described in the legend of Fig. 2, and see also the text for

further details.

Review article S. Glanz and U. Kuck

Rat1 and Rat2 (RNA maturation of psaA tscA RNA), both

of which are encoded by two adjacently located nuclear

genes, are also part of the maturation process. Interestingly,

only when both genes are simultaneously transferred into the

corresponding splicing-deficient mutant, they are able to

restore the wild-type phenotype. The deduced amino acid

sequence of Rat1, which directly interacts with tscA RNA,

shows 26% sequence homology to the conserved NADþ-

binding domain of poly(ADP-ribose) polymerases (PARP).(88)

All proteins involved in processing of the tscA precursor are

associated with the thylakoid membrane. The processed tscA

RNA is also associated with a stromal 1 700 kDa protein

complex that additionally contains the exon 1 primary

transcript with its 50-intron. A component of this protein

complex is Raa3, showing homologies to pyridoxamine 50-

phosphate oxidases. The cofractionation of these two RNAs

together with Raa3 was shown by size exclusion chromato-

graphy.(89) Recently, another factor (Raa4) that shares a small

protein domain with tRNA synthetases was shown to be

involved in splicing of the first group II intron, and it remains to

930

be determined whether it is also part of a high-molecular-

weight complex (Glanz, unpublished).

A biochemical approach including UV-crosslinking experi-

ments, yeast three-hybrid analysis and mass spectrometry

identified three further chloroplast proteins with a more

general affinity to group II introns. These include a 31 kDa

protein with a 39% sequence homology to the NADþ-binding

domain of 6-phosphogluconate dehydrogenases Cpn60, a

bacterial homologue of GroEL ATPases, and a chloroplast-

localised cNAPL protein, showing high similarity to nucleo-

some assembly proteins.(83,90,91)

For splicing of the second intron, apparently two

membrane-associated complexes are involved (Fig. 6). The

first is the 670 kDa complex containing the above-mentioned

Raa1, together with so far uncharacterised RNA molecules

and protein factors. The second is a 400–500 kDa multiprotein

Raa1/Raa2 complex, which is probably not associated with

RNA, since no direct interaction with psaA-i2 or solubilised

chloroplast extracts in vitro was detected.(87) The Raa2

polypeptide contains conserved motifs with significant

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

Glossary

Autocatalytic splicing: Self-splicing of group I, II and III

introns in vitro under non-physiological reaction conditions

in the absence of protein factors. These introns are

referred to as ribozymes (ribonucleic acid enzymes) and

can catalyse their own cleavage or the cleavage of other

RNAs. However, efficient in vivo splicing almost always

requires the assistance of a catalytic enzyme, RNA

molecules and/or other protein factors that are either

encoded by the nucleus or the intron itself (maturases).

Mobile group II introns: Mobile group II introns are

found in bacterial and organelle genomes. They are both

catalytic RNAs and retrotransposable elements with an

intron-encoded protein that has reverse transcriptase

activity. Group II introns can transpose with high

efficiencies (retrohoming) into defined sites or can invade

at ectopic sites (retrotransposition).

Nuclear spliceosome: Nuclear pre-mRNA introns are

not able to splice autocatalytically without the assistance

of trans-acting RNA or protein factors. Eukaryotic pre-

mRNA splicing takes place in the spliceosome, a

ribonucleoprotein (RNP) complex of �60S that assembles

from the five U-rich small nuclear ribonucleoproteins

(snRNPs) U1, U2, U4, U5 and U6, which are temporarily

associated with more than 70 proteins such as RNA

helicases and SR proteins. For accurate spliceosome

assembly, a range of dynamic protein-protein, RNA-

protein and RNA-RNA interactions are required.

Splicing mechanism of group II introns: Splicing

S. Glanz and U. Kuck Review article

sequence similarity to two domains of pseudouridine

synthases; however, this enzyme activity is not a prerequisite

for trans-splicing. Therefore, Raa2 was speculated to be a

bifunctional protein acting in pseudouridination as well as

trans-splicing.(92) Moreover, it was suggested that this

complex represents a pre-spliceosome, which is assembled

and/or stabilised via three genetically defined factors. It was

discussed that this complex has an indirect role in recognition

and assembly of primary exon 2 and 3 RNAs, and thus this

complex may be involved in the storage of trans-splicing

factors. Finally, upon gene activation, this complex may

specifically be redistributed to the site of transcription.(87,92,93)

The spatial separation of the complexes into membrane

and stromal chloroplast fractions indicates that they may act in

different modes and at different steps in the psaA trans-

splicing process. The first reaction probably takes place in the

stromal phase, whereas the second reaction is associated

with the membrane. It can be further speculated that

membranous splicing of the second intron is coupled with

the translation and integration of the psaA protein into the

thylakoid membrane system.(87)

Although no homologues of C. reinhardtii factors have been

identified in higher plant chloroplasts (see Table 3), it may be

envisioned that proteins promoting trans-splicing act as RNA

chaperones and stabilise or support the correct folding of intron

structures. Alternatively, they may mediate splicing indirectly by

interaction with other protein factors. Indeed, cpRNPs in

tobacco were shown to act as stabilising factors for a number of

non-ribosome-bound stromal chloroplast mRNAs.(94) Even

though the exact functions of so far characterised factors in the

trans-splicing process have to be elucidated, the presented

high-molecular RNP complexes and splicing factors provide a

basis for the isolation and characterisation of further trans-

splicing factors and for the analyses of their general functional

role in an organelle spliceosome.

occurs via two sequential transesterification reactions.

First, the 20OH of a specific branch point nucleotide within

the intron performs a nucleophilic attack on the first

nucleotide of the intron at the 50-splice site forming the

lariat intermediate. Second, the 30OH of the released

50-exon performs a nucleophilic attack at the last

nucleotide of the intron at the 30-splice site, thereby joining

the exons and releasing the intron lariat.

Conclusions

Trans-splicing of discontinuous group II introns is a

phenomenon that occurs in a huge number of organelles

from plants and diverse lower eukaryotes. We provide a

complete survey of 187 organelle trans-spliced introns that

were predicted from the complete sequencing data of 179

organelle genomes. Furthermore, a summary of trans-

splicing factors that are supposed to promote group II intron

splicing is given. Genetic and biochemical data from splicing-

deficient mutants support the assumption that intron trans-

splicing is promoted by a set of trans-acting factors as part of

high-molecular-weight complexes. The presented model

predicts that these complexes may be involved in the storage

of trans-splicing factors and can specifically be redistributed

to the site of transcription. A spatial separation of the complex

BioEssays 31:921–934, � 2009 Wiley Periodicals, Inc.

in membrane and non-membrane fractions indicates further

different modes of action during the trans-splicing process.

Supporting Information available online

Acknowledgments: The experimented work of the authors is

supported by the Deutsche Forschungsgemeinschaft

(SFB480, B3).

931

Review article S. Glanz and U. Kuck

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