Trans-splicing mutants of <i>Chlamydomonas reinhardtii</i>

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Reference

Trans-splicing mutants of Chlamydomonas reinhardtii

GOLDSCHMIDT-CLERMONT, Michel P., et al.

Abstract

In Chlamydomonas reinhardtii the three exons of the psaA gene are widely scattered on the chloroplast genome: exons 1 and 2 are in opposite orientations and distant from each other and from exon 3. The mature mRNA, encoding a core polypeptide of photosystem I, is thus probably assembled from separate precursors by splicing in trans. We have isolated and characterized a set of mutants that are deficient in the maturation of psaA mRNA. The mutants belong to 14 nuclear complementation groups and one chloroplast locus that are required for the assembly of psaA mRNA. The chloroplast locus, tscA, is remote from any of the exons and must encode a factor required in trans. The mutants all show one of only three phenotypes that correspond to defects in one or other or both of the joining reactions. These phenotypes, and those of double mutants, are consistent with the existence of two alternative splicing pathways.

GOLDSCHMIDT-CLERMONT, Michel P., et al . Trans-splicing mutants of Chlamydomonas reinhardtii . Molecular and General Genetics , 1990, vol. 223, no. 3, p. 417-425

DOI : 10.1007/BF00264448 PMID : 2270082

Available at:

http://archive-ouverte.unige.ch/unige:143389

Disclaimer: layout of this document may differ from the published version.

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Trans-splicing mutants of Chlamydomonas reinhardtii

Michel Goldschmidt-Clermont 1, Jacqueline Girard-Bascou 2, Yves Choquet 1' 2, and Jean-David Rochaix ~

1 Departments of Plant and of Molecular Biology, University of Geneva, Sciences II, 30, quai E. Ansermet, CH-1211 Gen~ve 4, Switzerland 2 Institut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, F-75005 Paris, France

Received March 19, 1990

Summary. In Chlamydomonas reinhardtii the three exons of the psaA gene are widely scattered on the chloroplast genome: exons 1 and 2 are in opposite orientations and distant from each other and from exon 3. The mature mRNA, encoding a core polypeptide of photosystem I, is thus probably assembled from separate precursors by splicing in trans. We have isolated and characterized a set of mutants that are deficient in the maturation of psaA mRNA. The mutants belong to 14 nuclear complementation groups and one chloroplast locus that are required for the assembly of psaA mRNA. The chloroplast locus, tscA, is remote from any of the exons and must encode a factor required in trans. The mutants all show one of only three phenotypes that correspond to defects in one or other or both of the joining reactions.

These phenotypes, and those of double mutants, are consistent with the existence of two alternative splicing pathways.

Key words: Chloroplast - Photosystem I - RNA processing - Intron

Introduction

The psaA gene in the Chlamydomonas reinhardtii chloroplast has an unusual structure: it is formed of three exons that are widely separated on the circular genome and are transcribed from opposite strands (Kfick et al.

1987; Choquet et al. 1988). Exon i encodes 30 amino acid residues, and is 50 kb from exon 2, which encodes 60 residues but is transcribed from the other strand.

Exon 3 encodes 661 residues and is on the other side of the inverted repeat, some 90 kb away. Thus an unusual process must be required to allow expression of the psaA mRNA. The intron sequences at the borders of the exons follow the consensus of group II introns, and the two split parts of intron 2 can theoretically be folded into a secondary structure that conforms to the model Offprint requests to: M. Goldschmidt-Clermont

for this class of introns (Michel and Dujon 1983). The maturation of the transcripts is thought to involve the trans-splicing of separate precursors of the three exons (Choquet et al. 1988; Herrin and Schmidt 1988). The precursor of exon 2 also contains the sequence ofpsbD, coding for the D2 protein ofphotosystem II. The expression of the psaA gene is, however, not significantly af- fected in a mutant that lacks stable psbD mRNA, sug- gesting that trans-splicing can occur before the psbD sequences are degraded (Kuchka et al. 1989): in this case at least, the accumulation of the two mRNAs is not tightly coupled.

The psaA gene encodes one of two related polypeptides (84 and 82 kDa) that are the apoproteins of CPI, the chlorophyll complex containing the reaction center chlorophyll P700 and the first electron acceptors of photosystem I (PSI). The gene for the other related polypeptide, psaB, has a normal continuous structure (Kfick et al. 1987). Both psaA and psaB are also continuous genes in the chloroplast genomes of other plants and of cyanobacteria where they are adjacent and co-transcribed (Fish et al. 1985; Kitsch et al. 1986; Lehmbeck et al. 1986; Ohyama et al. 1986; Shinozaki et al. 1986;

Cantrell and Bryant 1987). The two genes in Euglena gracilis are also adjacent, but they are split by three and six introns, respectively, in positions that differ from the interruptions in the C. reinhardtii psaA gene (Cush- man et al. 1988).

Trans-splicing is an intriguing complication of gene expression that also operates in other organisms. The rpsl2 mRNAs in the chloroplasts of tobacco, rice and of the liverwort Marchantia polymorpha are probably also assembled in trans from two separate precursors (Torazawa etal. 1986; Fukuzawa etal. 1986; Koller et al. 1987; Zaita et al. 1987; Kohchi et al. 1988; Hirat- suka et al. 1989). A related situation prevails in trypanosomes and other Kinetoplastida where the same short common leader is spliced in trans to the body of most or all of the mRNAs (reviewed by Laird 1989). In the nematode Caenorhabditis elegans certain mRNAs also share a common leader which may be added in a trans- splicing reaction (Krause and Hirsh 1987).

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418

Trans-splicing

places additional demands on the splicing machinery. In contrast to the usual intramolecu- lar

cis-splicing

of other pre-mRNAs,

trans-splicing

re- quires a bimolecular reaction between separate exon precursors. In the chloroplast of

C. reinhardtii,

a further requirement is for specificity in the selection of the appropriate pairs of precursors to avoid the generation of RNAs with scrambled exons. It would be of interest to understand how these reactions occur, what factors are required, what the possible implications are for the regulation of gene expression and how such a complex situation has evolved. In order to gain information relevant to some of these problems, we have isolated and analyzed a large number of

C. reinhardtii

mutants that have defects in the maturation of

psaA

mRNA. The mutants reveal the contributions of an unexpectedly large number of nuclear genes and of a chloroplast gene that are all required for

psaA trans-splicing.

They accumulate abnormal transcripts that provide information on the pathways

ofpsaA

mRNA maturation.

Materials and methods

Strains and growth conditions. C. reinhardtii

wild-type strain 137c was used in this work. Thirteen nuclear PSI mutants, including F19, M18, F31, M10 and F15 were previously described (Girard et al. 1980). Nine nuclear mutant strains, including Fl38, Fl55 and Fl25 were obtained from Dr. J. Gamier (Gamier et al. 1979). The chloroplast mutant Fud3 was previously described (Gir- ard-Bascou 1987; Girard-Bascou et al. 1987).

General handling procedures and growth media (TRIS-acetate-phosphate, TAP; high salt minimal, HSM) are described by Harris (1989). The strains were grown at 25 ° C in TAP medium, photosynthetic mutants were maintained in reduced light (200-300 lx).

Mutagenesis.

The mutants were obtained after treatment of the wild-type strain 137c mt(+) with 5-fluoro-deoxy- uridine (strains Ll18, L135F, L121G, L133B, L135F, LI36F, L137G, L138A were mutagenized with UV light), followed by an enrichment step in the presence of metronidazole according to Bennoun and Delepelaire (1982). PSI mutants were identified by their fluorescence yield, which is approximately threefold higher than wild type, and by their fluorescence induction kinetics which display no decrease phase (Bennoun and Chua 1976;

Girard et al. 1980). Only mutants of a given phenotype which were isolated in different experiments or from separate flasks were retained so as to ensure that they arose independently.

Segregation analysis.

Gametogenesis was induced by transferring exponentially growing cells to TAP agar medium containing only 0.04 g/1 NH~C1 for 3 days under low light (200-300 lx). Alternatively, gametogenesis was obtained in TAP medium without NH~C1 after 1 day.

The gametes were mixed and after 3 h the mating sus- pension was spotted on TAP 3% agar or HSM 4% agar plates. Maturation of zygotes and dissection of the te-

trads were carried out essentially according to Levine and Ebersold (1960).

The mode of inheritance was investigated in crosses between the PS I mutant mr(+) and the wild-type mt(-). Individual colonies derived from each meiotic product were characterized by their fluorescence induction kinetics as described above. While nuclear markers are inherited with Mendelian segregation patterns (2 mutant: 2 wild type), chloroplast markers are inherited uniparentally from the rot(+) parent in most tetrads (4:0).

At least ten tetrads were scored in each cross.

Recombination tests between the nuclear PSI mutants involved either tetrad analysis as above or zygote clone analysis. For the latter, clones derived from individual zygotes were resuspended in phosphate buffer, spotted on HSM agar plates and grown in the light to detect wild-type recombinants.

Complementation tests.

After gametogenesis, gametes of opposite mating types in phosphate buffer (10mM, pH 7) at a density of approximately 5 x 10 7 cells/ml, were mixed in small petri dishes (3.5 cm) in a final vol- ume of 1.4 ml in the presence or absence of 100 gg/ml chloramphenicol. At 1-3 h after mating, zygotes aggre- gate and form a continuous pellicle at the surface and on the bottom of the dish. In all the crosses examined, a high efficiency of mating was obtained. After 48 h the zygote pellicles were washed with phosphate buffer (containing chloramphenicol when appropriate) to elimi- nate unmated gametes and fluorescence measurements were performed directly on the plate. All crosses were repeated at least twice.

RNA analysis.

In the initial screening, total RNA was obtained from 10 ml cultures at a density of 2-4 x 106 cells/ml using the following mini-preparation proce- dure. Cells were collected by centrifugation (6000 x g, 5 min), resuspended in 1.5 ml 10 mM TRIS-HC1 pH 7.4 on ice and transferred to a 2.2 ml microcentrifuge tube, centrifuged briefly (13000 g, 30 s) and in some cases stored frozen. To the (frozen) pellets was added 0.6 ml TEN-SDS (0.2 M TRIS-HC1, pH 8, 0.5 M NaC1, 0.01 M EDTA, 0.2% SDS) and 0.6 ml phenol:chloroform:isoa- mylalcohol (25:25:1), and the cells were immediately resuspended and lysed by sonication. After centrifugation (13 000 x g, 1 min) the supernatant was transferred to a new 2.2 ml microfuge tube and 1.4 ml ethanol was added. The RNA was precipitated at - 2 0 ° C, collected by centrifugation (13 000 x g, 5 min), washed with 70%

ethanol, dried briefly, dissolved in 0.1 ml of 5 mM EDTA, pH 7.5 and treated with approximately 0.2 gl diethyl pyrocarbonate to inactivate any residual RNAse.

Large-scale RNA preparations followed the CsC1 proce- dure described previously (Rochaix et al. 1988). Gel electrophoresis and blotting were as described (Gold- schmidt-Clermont 1986). The 32P-labelled probes for exon 1 (fragment H 0.4 of R21 subcloned in pUC19), exon 2 (oligonucleotide 502) and exon 3 (fragment HR 2.2 of R17 subcloned in pBR325) were labelled and hybridized as before (Choquet et al. 1988).

Total DNA was prepared (Rochaix et al. 1988), di-

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gested with restriction enzymes, subjected to electrophoresis, blotted to nitrocellulose and hybridized with restriction fragments of cloned DNA labelled by nick- translation (Goldschmidt-Clermont 1986). The filters were additionally washed in 1 mM EDTA, 0.01% SDS at 65 ° C. This reduced the signal, but improved the specificity of hybridization with the R16 RH1.4 probe, which contains repetitive DNA.

Results

Isolation ofpsaA mRNA maturation mutants

We initially screened a collection of photosystem I mutants that had been previously characterized (Girard et al. 1980; Girard-Bascou 1987). Total RNA was ex- tracted from mutants representative of 13 nuclear complementation groups and two chloroplast loci. Northern analysis showed that mutants in five of the nuclear complementation groups and one of the chloroplast loci failed to accumulate normal psaA mRNA but all had an identical pattern of new bands. These mutants in different nuclear and chloroplast loci thus shared an identical phenotype in the Northern analysis, which we subsequently called class C. In order to extend this analysis and identify other possible phenotypes, we generat- ed a large set of additional PSI mutations. Colonies were screened first for high fluorescence and then for the fluorescence induction kinetics typical of defects in PSI. Using a rapid RNA extraction protocol, we then screened these PSI mutants for the presence of unusual psaA transcripts. Among 39 independent mutants tested, we found 17 that were deficient in psaA mRNA maturation. They showed three types of phenotypes (Fig. 1):

the class C pattern of transcripts was again found in eight mutants. Two additional patterns were also observed, defining classes A (six mutants) and B (three mutants). Mutants in class A fail to assemble exons 2 and 3, mutants in class C fail to assemble exons ] and 2 and those in class B are blocked in both steps (Choquet et al.

1988). We also analyzed nine other PSI mutants described by Garnier et al. (1979): seven of them had abnormal psaA transcripts. One belonged to class A, three to class B and three to class C.

The fluorescence induction kinetics in mutants that are deficient in both PS I and PS II resemble those of the PS II mutants (i.e. PS II mutations are epistatic to PSI mutations for the fluorescence induction phenotype). We therefore also screened a collection of 24 ap- parent PS II mutants for abnormal psaA transcripts and found 2 that had the class C pattern of transcripts (L237A and L210A). These two mutants also lacked the psbA mRNA completely, a deficiency that could ac- count for their PS II phenotype (data not shown).

While nuclear markers are inherited with Mendelian segregation patterns, chloroplast markers are inherited uniparentally from the mt(+) parent in C. reinhardtii.

Most of the mutations showed 2: 2 segregation in a cross to the wildtype and were thus localized to the nuclear genome. In addition to Fud3 (Girard-Bascou 1987), two

0 EXON 1

I 234 5678 9

b EXON 3

I 2 3 z, 5 6 7 8 9

o Jnhmm"m~,l

• ) ) ) ) •

A B A B C C C C WT A B A B C C C CWT Fig. 1. Phenotypes of the psaA mutants. Total RNA from a set of mutants was separated by gel electrophoresis, blotted to nitrocellulose and hybridized to probes specific for exon 1 (panel a) or exon 3 (panel b). The three classes of phenotypes (A, B or C) are indicated at the bottom of each lane. The bands are identified by symbols that refer to the putative precursors or intermediates as in Fig. 4. Lane 1, L136F; lane 2, Ll18; lane 3, LI21G; lane 4, L137H; lane 5, L133B; lane6, L135F; lane7, L137G; lane8, L138A; lane 9, wild-type (wt)

other new mutants from class C (H13 and D42) and the two class C mutants with defects in both PS II and PSI (L237A and L210A) showed uniparental inheritance of their deficiencies and were ascribed to the chloroplast genome.

Complementation analysis of nuclear mutants

The nuclear mutants were further analyzed in complementation tests to determine whether they affect the same or different genes (Table 1). This can be achieved by mating two different PSI mutants and testing the fluorescence induction kinetics of the resulting diploid zygotes (Bennoun et al. 1980), as illustrated in Fig. 2.

Zygotes with mutations that complement (panels b, c) have fluorescence curves resembling the wild-type (panel a): such mutations most probably alter different genes (although we have not ruled out the possibility of intracistronic complementation). Zygotes with two non-complementing mutations (panel d) have fluorescence curves that are identical to those of the mutant parents or the homozygotes. Complementation is completely inhibited by the addition of chloramphenicol which prevents chloroplast protein synthesis during zygote formation. The chloramphenicol controls show that the complementing phenotypes are not due to the presence of revertants in the cultures used for the crosses.

Failure to complement occurs if two mutations affect the same gene, but could also be due to a defect in zygote maturation. As a control in the cases where two mutations failed to complement, their linkage was deter- mined by measuring the frequency of wild-type recom-

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Table 1. Complementation table Class A

F138 HN12 HN22 LI36F LI21G HN54 L8

(-) (-) (-) (-) (-) (-) (-)"

+ + + + + nt

+ + + + nt

- + + nt

+ + nt

+ nt

nt F138

(+)

HN12 +

(+)

HN22 + +

(+)

L136F + + --

(+)

L121G + + + +

(+)

HN54 + + + + +

(+)

L8 + + + + + -

(+)

Class B

HN31 F155 LI37H L118

(-) (-) (-) (-)

HN31 -- -- +

(+)

F155 - - +

(+)

L137H -- -- +

(+)

LII8 + + +

(+)

Class C

F19 MI8 F31 MI0 F15 F125 HN63 L135F

(-) (-) (-) (-) (-) (-) (-) (-)

F125 + + + + + - +

(+)

HN63 + + + + + - +

(+)

L135F + + + + + + +

(+)

" The L8(-) strain mated poorly and could not be tested (nt) The fluorescence induction kinetics of zygotes were scored 48 h after mating. Complementation (+) gave rise to kinetics resembling the wild type, but to mutant curves in the presence of chloramphenicol (reversion control). Failure to complement (--) gave rise to mutant curves in both cases. For class C, three new mutants (F125, HN23, L135F) were tested for complementation together with representatives of five complementation groups previously defined (Girard et al. 1980)

2D

%

Ill o C 0J o gl

P o LI.

t3

PSI mutant

S

I I i I

L118 (*) x Ft55 (-)

÷Cm -Crn

L~IS(+) x HN31(-)

÷Cm

b

HN31(+) x FI55(-)

*Cm

(~ -Cm

I d I I I I

; o'5 1 1; 0 0.s 1 1.s

Time (s)

Fig. 2. Comptementation analysis. Mutants from class B were crossed as indicated in panels b, c and d in the absence (-Cm) or in the presence of chloramphenicol (+ Cm). After 48 h the zygotes were washed and analyzed. The fluorescence induction curves typical of the wild-type and of photosystem I mutants (note the absence of a decrease phase) are shown in panel a. Zygotes with mutations that complement are shown in panels b and c; complementation is blocked by the addition of chloramphenicol (+ Cm).

The mutations in the zygotes in panel d fail to complement

Table 2. Recombination analysis

Cross Number of tetrads Number of zygotic

scored clones

Class A

L8 x HN54 11 23

L136 x HN22 28 NT a

Class B

HN31 x F155 12 24

HN31 x L137H 6 26

F155 x L137H 18 22

Class C

F125 x HN63 22 30

a The reversion frequency of HN22 was high and interfered with this test. NT, not tested

Mutations that fail to complement (Table 1) probably affect the same gene and should be closely linked. Zygotes from the corresponding crosses were dissected for tetrad analysis, and the fluorescence induction kinetics of the progeny were scored. All tetrads tested showed 4:0 segregation of the mutant phenotype. Alterna- tively, zygotic clones were scored for the presence of wild-type recombinants by spotting drops on minimal medium. Of all the zygotic clones that were tested, none gave rise to wild-type progeny

binants in the meiotic progeny of the zygotes: if two mutations alter the same gene they must be closely linked. In all cases the recombination frequency was very low (Table 2), as expected for mutation that affect the same gene. The possibility of dominant alleles, which

might also fail to complement in this analysis, can be ruled out because all mutations do complement members of at least one other group.

The results of this complementation and recombination analysis are summarized in Tables 1 and 2. Class A

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has five different complementation groups defined by seven mutants: three groups are represented by one allele and two by two alleles. The four mutants in class B belong to two groups with one and three alleles, respectively. Of the many new nuclear mutants in class C, three were analyzed with respect to representatives of five complementation groups that were described previously (Girard et al. 1980), defining two new groups.

In order to simplify this analysis, we have treated the three phenotypic classes separately, making the assumption that mutations with different phenotypes belong to different complementation groups. In six cases where members of different classes were crossed (see double mutants below), the mutations segregated readi- ly, showing that the assumption is probably valid in most cases.

It is clear that we have not identified all the genes that are involved in psaA m R N A maturation: the loci in classes A and C are far from being saturated since six of them are represented by only one allele. There may be fewer genes in class B, where four mutants define two complementation groups. There are thus probably more than 14 nuclear genes required for the maturation of psaA mRNA.

Chloroplast mutations

The five chloroplast mutants (Fud3, H13, D42, L210A, L237A) that were defective in psaA m R N A maturation had the class C phenotype. In addition they were also all yellow when grown in the dark, in contrast to wild- type C. reinhardtii which is normally green in the dark.

This alteration was also inherited uniparentally and in the case of Fud3, it co-segregated with the P S I defect in biparental zygotes (Girard-Bascou 1987). This sug- gested that the mutational lesions might affect both psaA m R N A splicing and chlorophyll synthesis in the dark, and could be due to large D N A alterations affecting several chloroplast genes. An analysis of chloroplast D N A from the five mutants indeed showed that they lack a region of the plastid genome corresponding to a part of fragment R12 (Fig. 3) and extending into the adjacent fragment R16. Roitgrund and Mets (1990) have independently obtained and described similar mutants.

In the two mutants that also have an additional defect in PS II and lack psbA m R N A (L237A and L210A), the deletions extend into the adjacent inverted repeat (Fig. 3). The other copy of the inverted repeat is also partly deleted (fragment R14), so that the mutants lack both copies of the psbA gene. Because all these deletions result in the class C phenotype, and affect the same region far from any of the exons ofpsaA, they probably disrupt a function that is required in trans for the trans- splicing of exons 1 and 2 and define a genetic locus called tscA (trans-splicing chloroplast). Deletions such as FUD7 (Bennoun et al. 1986), which remove psbA but have no effect on psaA trans-splicing (unpublished data) do not affect the tscA locus (Fig. 3). The basis of the associated yellow-in-the-dark phenotype which also maps to this region will be described elsewhere.

Q

psbD---~ ~

l/

tsc" ~k~"~. / 7 p s ~ B ~ p s ~ A - ~

b KS 1.0 RH 1.4

EcoRI _, PstI _, EcoRI _{r - - ~}Hindlll

< ~

¢~ f- - -~_~ o ¢,~ ;~ ~ ~

RP 3.6

I>

RP I.~ RH 1.4

•

C

psbA + tscA + psbA + tscA-

R12 R16

~ ~ ,~

I KS~.O I I RH~.4 I

R P H R H

<i RPI.4--~ < RP 3,6 ) ~--RHI.4--~

,,~==FUD3,H13 oo=

H I IWT

psbA- tscA- ,=o= L210A. L237A

psbA- tscA + ... FUD 7

tscA psbA

,~oo, ,~== _, _/1=;>

Fig. 3 a-e. Mapping of the tscA deletions, a A schematic representa- tion of the Chlamydomonas reinhardtii chloroplast genome. The outer circle shows the positions of the two copies of the inverted repeat (black bars) and of a few genes relevant to this work (open bars, not drawn to scale). The inner circle shows the relevant EcoRI fragments, b Southern analysis of chloroplast mutants. Total DNA from the wild-type (WT) and from two chloroplast mutants (HI 3, L210A) was digested with EcoRI and PstI (left) or EcoRI and HindIII (right), subjected to agarose gel electrophoresis, blotted to nitrocellulose and hybridized with the 32p-labelled DNA fragments KS1.0 (left) and RH1.4 (right). These probes, and the wild- type fragments to which they hybridize, are shown in e. The novel junction fragments in the mutants are indicated with triangles. The black triangle points to a doublet in H13 which contains the novel fragment and the wild-type fragment from the other copy of the inverted repeat (Y. Choquet, unpublished). Both fragments are missing in L210A. Minor bands are due to the cross-hybridization of chloroplast repetitive DNA (right) or to slightly incomplete di- gestion (left). e Physical map of the tscA locus. The wild-type DNA is represented by the upper bar with the inverted repeat in black and some relevant restridtion sites (R, EcoRI; P, PstI; H, HindIII).

The lines at the top show the 1.0 kb KpnI- Sinai fragment from R12 (KS1.0) and the 1.4 kb EcoRI--HindIII fragment from R16 (RH1.4) that were used as hybridization probes in b. The approxi- mate extent of the deletions in four chloroplast mutants is shown by the gaps in the respective bars. The psbA mutant FUD7 (Ben- noun et al. 1986) is also depicted for comparison. The phenotypes of the various strains are indicated on the left. L210A, L237A and FUD7 are also deleted for the other copy of psbA in the inverted repeat (fragment R14, not shown)

Alternating trans-splicing pathways

We have found that the psaA m R N A mutants belong to three different classes with distinct patterns of transcripts. A detailed molecular characterization of these transcripts (Choquet et al. 1988) has shown that class A

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422

WILD-TYPE FF1- IT], ~ o

El 121 3 14

CLASS A I-T], l~q, ~ o

I~ [.. j 1

-~.- ~

, 1

~ * _I. _~

i

-. .

"'r"

I

CLASS B CLASS C

[3-1, IZI° ~ o

t ~ L )

"-.¥/" . -y- .-

~ ~

[5i5- [2~, ~-~--~°

~1 I ~

~ . .

,, Y

' 121 3 Io

L . . J

. .

Y

Fig. 4. Trans-splicing scheme. The three exons are transcribed as separate precursors: exon 1 (I, 0.4 kb), exon 2 (~, cotranscribed with psbD, 3.6 and 7 kb transcripts differing in their 3' ends downstream of exon 2), exon 3 (o, 2.4 kb). Intermediates are forined from the precursors which are trans-spliced, beginning either with exons 1 and 2 (., exonl-exon2, 2.6 and 6 kb intermediates differing in their 3' ends) or with exons 2 and 3 (~, psbD-exon2-exon3, 3.8 kb). These are in turn trans-spliced to the remaining exon to form maturepsaA mRNA (~, 2.8 kb). In class A mutants, exons 2 and 3 cannot be joined (dotted lines) and the exonl-exon2 intermediates (.) accumulate. In class C mutants, exons 1 and 2 are not joined, and the psbD-exon2-exon3 intermediate (~) accumulates.

In class B, the two initial steps are blocked, and the three separate precursors are found. (For a detailed analysis of the various transcripts, see Choquet et al. 1988)

AxC BxC

Parents TetrQtype Tetratype

WT A B C I 2 3 4 1 2 3 4

• •

= :

Fig. 5. Tetrad analysis of double-mutant phenotypes. Left panel.

Total RNA from wild-type (WT) and the three parent strains (A, L136F; B, Ll18; C, LI35F) was subjected to gel electrophoresis, blotted to nitrocellulose and hybridized with a probe specific for exon 2. The bands are labelled with symbols that refer to the transcripts as diagrammed in Fig. 4: mature RNA (.~, 2.8 kb); psbD- exon2-exon3 (~, 3.8 kb); psbD-exon2 (i, 3.6 and 7 kb, the minor 7 kb transcript extends further downstream of exon 2); exonl- exon2 (., 2.6 and 6 kb, the minor 6 kb intermediate extends further downstream of exon 2 and is probably derived from the minor psbD-exon2 precursor). Center panel. Total RNA from the four progeny of a tetratype tetrad of the A × C cross, analyzed as in the left panel. Lane 1, A; C double mutant showing the B phenotype; lane 2, A mutant; lane 3, C mutant; lane 4, wild type. Right panel. The four progeny of a tetratype tetrad from the B x C cross.

Lane 1, wild type; lanes 2, 3, B mutant and B; C double mutant with the B phenotype; lane 4, C mutant

mutants accumulate RNA molecules with exons 1 and 2, but not exons 2 and 3, properly spliced, (Fig. 4). Con- versely, class C mutants have transcripts with exons 2 and 3 properly spliced but not exons 1 and 2. Class B mutants have transcripts that only contain individual psaA exons and no spliced intermediates. It thus appears that two alternative splicing pathways are possible, beginning with either the splicing of exons i and 2 or the splicing of exons 2 and 3. If there is indeed no required order to the splicing reactions, the mutations are not expected to have a simple epistatic relationship, as in linear pathways where mutations upstream mask the effect of mutations downstream. Instead, the model (Fig. 4) predicts that all three combinations of double mutants from the three different phenotypic classes (A;

B, A; C, or B; C) will have the phenotype of class B.

This hypothesis was tested by analysing tetrads from crosses of mutants representing the different classes (Fig. 5, left panel). A tetratype tetrad from a cross between members of class A and class C (center panel) consists of one wild-type (lane 4), and three mutant progeny: one with the mutation of the class A parent (lane 2), one with the mutation of the class C parent (lane 3), and one double mutant. As predicted from the model, this A; C double mutant does have the phenotype

of class B (lane 1). Similarly, in a tetratype from a cross between representatives of classes B and C (Fig. 5, right panel), we find one wild-type progeny (lane 1), one with the mutation of class C (lane 4), and two which have the phenotype of class B (lanes 2, 3): one of the latter must carry the B mutation, while the other must be the double mutant (B; C). A similar result was obtained in the analysis of the progeny of a cross between mutants of classes A and B: the double mutant (A; B) also had the phenotype of class B (not shown). The results were confirmed by analysing six additional double mutants in non-parental ditype tetrads using two different mutants for each of the three classes (data not shown).

The proposed scheme (Fig. 4) implies that the mutations in classes A and C affect factors which are required irrespective of whether exon 2 is present in its free form or is already spliced. This is confirmed by the double mutants : in class A for example, exon 3 is spliced neither to the intermediate containing exon 2 already spliced to exon 1 (in an A mutant) nor to the precursor of exon 2 (in an A; C double mutant). We cannot rule out that there might be additional factors which are specifically required for only one of these steps, but mutations affecting them would not be observed because of the re- dundancy in the splicing pathways.

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Discussion

Numerous nuclear genes are required for chloroplast

psaA

trans-splicing

We have screened a collection of

C. reinhardtii

mutants for defects in the maturation of

psaA

mRNA. A large proportion of the mutants we analyzed have defects in

trans-splicing

and accumulate abnormal transcripts. In the Northern analysis, they all show one of three different patterns of new

psaA

RNA bands and are ascribed on this basis to three phenotypic classes. A complemen- ration analysis shows that there are many genes in each of these three classes. We have assumed that mutations in different classes affect different genes. Although we have not completely ruled out the possibility that some mutations with different phenotypes may belong to the same complementation group, it is certainly not the general rule: in six different crosses, mutations from different classes segregated freely. Many of the 14 complemen- ration groups that were identified are represented by only one allele so it is reasonable to expect that additional genes have yet to be identified. Consequently there are probably many more than 14 nuclear genes specifically involved in the expression of just a single chloroplast gene.

This specificity is somewhat surprising but it seems to be the general rule: other nuclear genes are also specifically required for the expression of individual chloroplast genes in

C. reinhardtii.

Examples include the two nuclear loci,

NAC2

and

NAC1,

which are involved in the stabilisation of the

psbD

mRNA and the translation or degradation of the D2 polypeptide (Kuchka et al.

1988, 1989). Mutations in two nuclear genes prevent translation of the

psbC

mRNA (Rochaix et al. 1989) and another mutation blocks the accumulation

ofpsbB

mRNA (Jensen et al. 1986). The expression of chloroplast genes thus appears to depend on the contribution of multiple nuclear loci at many post-transcriptional steps controlling gene expression: RNA splicing, mRNA stability and translation. These genetic approaches have led to the identification of nuclear genes that are required for the activity of single chloroplast genes. There are probably also genes that are required for the expression of many chloroplast genes, but by screening for chlorophyll fluorescence mutants we have selected for mutations causing specific defects in photosynthesis rather than pleiotropic alterations of chloroplast gene expression which might have a more severe or lethal phenotype.

The dependence of organellar gene expression on specific nuclear loci is not unique to the

Chlamydomonas

chloroplast: similar mutants have been described in higher plants (Leto et al. 1985; Barkan et al. 1986; Tay- lor 1989). A large contribution of nuclear genes to the expression of specific mitochondrial genes has also been found in yeast (reviewed by Attardi and Schatz 1988).

For the splicing of certain mitochondrial group I introns in fungi, a protein is required which has a dual function: as a splicing factor and also as a tRNA synthetase (Akins and Lambowitz 1987; Herbert et al. 1988).

The spliceosomes of eukaryotic nuclei are composed of many proteins but also of snRNAs (reviewed by Mania- tis and Reed 1987), and RNA processing enzymes some- times also contain an RNA subunit (reviewed by Cech and Bass 1986). It will be of interest to determine the biochemical nature and function of the many nuclear and chloroplast

trans-splicing

factors that we have genet- ically identified in

C. reinhardtii.

The trans-splicing pathways

The large number of genes required for

psaA

expression may reflect the complexity of

trans-splicing:

in addition to the need for splicing factors, there may also be a requirement for factors that expedite the recognition of the two separate intron moieties which have to interact in the bimolecular reactions. Mutants in class B splice neither exons 1 and 2 nor exons 2 and 3. The two corresponding gene products are thus required for the

trans-

splicing of both of the split introns. Transcripts of the

psbA

gene and of the 23 S rRNA gene which contain group I introns are apparently spliced normally in these mutant strains. We speculate that the class B genes encode factors required for

trans-splicing

or perhaps for group II intron splicing in general (to our knowledge no other group II introns have yet been identified in the

C. reinhardtii

chloroplast genome). Mutants in class A fail to splice exon 3 to exon 2, regardless of whether the latter is already spliced to exon 1. However they are able to splice exons 1 and 2. The five nuclear genes in class A might encode factors necessary for the splicing or recognition of the two split parts of intron 2 or of the adjacent exon sequences. Conversely, mutants in class C cannot splice exon 1 to exon 2, irrespective of whether the latter is already spliced to exon 3 or not, but they do splice exons 2 and 3 normally. The seven corresponding nuclear genes, as well as one chloroplast gene, might be required in the splicing or recognition of the two separate parts of intron 1 or of the neighbour- ing exon sequences.

In the mutants from the three classes, the

psaA

^transcripts that accumulate are apparently normal precursors and intermediates in the proposed

trans-splicing

pathways, and they behave as can be expected in double mutant combinations. We have not obtained any mutants with aberrant

trans-splicing

leading to the accumulation of abnormal molecules with scrambled or unspe- cific exons. Mutations of this type might have led to lethal phenotypes by interfering with other essential functions in the chloroplast. Alternatively, the specific recognition of the split introns and the selection of the appropriate exons may rely on the formation of exten- sive intermolecular secondary structures that cannot be altered by simple mutational changes.

The evolution of trans-splicing

During evolution, most of the genes in the endosymbiot- ic ancestor of the chloroplast have been transferred to

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424

the nucleus. As a consequence there is a high degree of interdependence in the coordinate functioning of the two compartments. We have found one locus in the chloroplast genome, but many more in the nucleus which are required to ensure the proper trans-splicing of the psaA mRNA. Although trans-splicing also occurs in land plants, it is in a different gene (rpsl2), which is continuous in C. reinhardtii (Liu et al. 1989). It might be speculated that trans-splicing may have evolved independently in these organisms in a process of convergent evolution, but the many nuclear genes that are required for the process would then also have had to evolve simul- taneously in order to meet a new requirement of chloroplast gene expression. Trans-splicing is perhaps more likely to represent an ancestral mechanism which has been retained only in a few instances (Laird 1989). The genes that are required for the process would then have been present in the endosymbiont and could have been transferred to the nucleus.

Acknowledgements. We thank Dr. Jacques Garnier for providing mutants, Frangoise Laquerri&e and Mich~le Schirmer-Rahire for their excellent technical assistance. We also thank O. Jenni and F. Bujard-Ebener for preparing the figures, and S. Purton for his comments on the manuscript. This work was supported by the Fonds National Suisse pour la Recherche Scientifique (number 3.328-086) and by the French CNRS (grant URA D 1187).

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Communicated by R.G. Herrmann