Photosynthesis Research 82: 301–314, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.
301
Review
Chloroplast RNA processing and stability
David L. Herrin1,* & Jöerg Nickelsen2
1
Section of Molecular Cell and Developmental Biology, 1 University Station A6700, University of Texas at
Austin, Austin, TX 78712, USA; 2Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität
Bochum, 44780 Bochum, Germany; *Author for correspondence (e-mail: [email protected]; fax: +1512-471-3843)
Received 26 November 2003; accepted in revised form 18 March 2004
Key words: Chlamydomonas, chloroplasts, group I introns, group II introns, light-regulated splicing,
nuclear–chloroplast interactions, nucleolytic RNA processing, RNA degradation, RNA stability, transacting factors
Abstract
Primary chloroplast transcripts are processed in a number of ways, including intron splicing, internal cleavage
of polycistronic RNAs, and endonucleolytic or exonucleolytic cleavages at the transcript termini. All chloroplast RNAs are also subject to degradation, although a curious feature of many chloroplast mRNAs is their
relative longevity. Some of these processes, e.g., psbA splicing and stability of a number of chloroplast
mRNAs, are regulated in response to light–dark cycles or nutrient availability. This review highlights recent
advances in our understanding of these processes in the model organism Chlamydomonas reinhardtii, focusing
on results since the extensive reviews published in 1998 [Herrin DL et al. 1998 (pp. 183–195), Nickelsen J 1998
(pp. 151–163), Stern DB and Drager RG 1998 (pp. 164–182), in Rochaix JD et al. (eds) The Molecular Biology
of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The
Netherlands]. We also allude to studies with other organisms, and to the potential impact of the Chlamydomonas genome project where appropriate.
Abbreviations: C. reinhardtii – Chlamydomonas reinhardtii; ORF – open reading frame; UTR – untranslated region
RNA splicing
RNA splicing has been of considerable interest to
molecular biologists since the discovery of split
adenoviral genes in 1977. Not long after this discovery, the first intron-containing chloroplast gene
was described in Chlamydomonas reinhardtii; the
23S large subunit rRNA gene (Rochaix and Malöe
1978). In addition to the evolutionary implications
of ‘genes in pieces’, RNA splicing has been of
considerable mechanistic interest, because of the
great precision required for correct RNA splicing.
Also, in some cases, it has proven to be a highly
regulated step in gene expression.
Introns in chloroplast genes of Chlamydomanas
reinhardtii
The completion of the chloroplast genome
sequence from C. reinhardtii has confirmed that
there are no other introns in the genome besides
those that are located in the psbA, psaA, and 23S
rRNA genes (Maul et al. 2002). These genes con-
302
Figure 1. Diagram of the Chlamydomonas reinhardtii psbA gene showing the four light-regulated group I introns (Cr.psbA1–Cr.psbA4).
The positions and direction of transcription initiation sites are indicated with arrows. The amino acid motifs (GIY-YIG and HNH)
found in certain homing endonucleases and mobile introns are indicated in the respective ORFs. The overall length of the gene is
6.8 kb.
tain two types of introns that are characteristic of
organellar introns in general, group I and group II
(Li and Herrin 2002). There are four group I
introns in the psbA gene (Cr.psbA1–Cr.psbA4)
(Figure 1), and one group I intron in the 23S large
subunit rRNA gene (Cr.LSU). These introns are
all much larger than typical nuclear mRNA
introns, ranging in size from 900 to 1800 bp.
Also, as indicated in Figure 1, three of the psbA
introns (Cr.psbA2–Cr.psbA4) have a large freestanding ORF (Holloway et al. 1999) as does the
Cr.LSU intron.
There are two group II introns in the psaA
gene, neither of which encode an ORF, but they
are atypical in structure and that they are spliced
in trans (reviewed in Herrin et al. 1998). Indeed,
the first psaA intron is dispersed among three
different RNAs, two containing the splice junctions and one completely internal to the intron;
this last RNA is encoded by the tscA gene
(Goldschmidt-Clermont et al. 1991). It is likely
that these introns splice only in a protein-dependent manner, as they are lacking some structures
found in self-splicing group II introns.
Thus, the intron composition of the C. reinhardtii chloroplast genome differs substantially
from that of angiosperms, where 17% of the
genes contain introns, and the vast majority are
cis-spliced group II introns (Plant and Gray 1988).
There is only one group I intron in the typical
angiosperm chloroplast genome; it is in the trnL
gene and appears to be an ancient intron inherited
from cyanobacteria (Kuhsel et al. 1990; Xu et al.
1990).
Analysis of species related to C. reinhardtii have
shown that group I introns are common in the
chloroplast genome of Chlamydomonas spp., particularly in the rRNA (Turmel et al. 1993) and
psbA (Turmel et al. 1989; O.W. Odom and D.L.
Herrin, unpublished results) genes. Evidence also
indicates that the trans-spliced psaA introns are
trans-spliced in other Chlamydomonas species,
suggesting that it is not a recently evolved process
(Turmel et al. 1995). Curiously, cis-spliced group
II introns have not been reported to date in the
chloroplast genome of Chlamydomonas spp.
However, one has recently been discovered in the
psbA gene of a psychrophilic Chlamydomonas
(O.W. Odom, D. Shenkenberg and D.L. Herrin,
unpublished results).
Group I introns: splicing mechanism and
self-splicing
All of the group I introns in C. reinhardtii selfsplice in vitro via the guanosine-dependent
mechanism first worked out for the Tetrahymena
rRNA intron (Cech 1990). Thus, these precursor
RNAs are also ribozymes. Biochemical requirements for efficient self-splicing of two of the
introns, Cr.psbA2 and Cr.LSU, respectively, were
examined in detail (Herrin et al. 1998), and the
rates of in vitro self-splicing at the optimum
temperature (45 C) were very high (kobs of
1 min)1), somewhat faster even than the in vivo
splicing rates of the psbA introns (Deshpande
et al. 1997). At the physiological temperature of
23 C, however, the rates of in vitro self-splicing
are significantly slower than in vivo splicing.
There is also the problem that these in vitro-
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synthesized pre-RNAs tend to have fast and
slow-reacting fractions. The size of the relatively
inactive fraction often increases with the length of
the pre-RNA, and can be due to intronic or
exonic sequences (Kuo and Herrin 2000). Taken
together with the extensive genetics of RNA
splicing in fungal mitochondria, it is probable
that proteins promote the splicing of these selfsplicing introns in vivo. Recently, suppressor
genetics was used in C. reinhardtii to identify
nuclear genes that promote splicing of the
Cr.LSU intron and at least one psbA intron (Li et
al. 2002, see below).
Light-dependent splicing of the psbA introns
Splicing of all four of the psbA introns is strongly
promoted by light in C. reinhardtii growing
photoautotrophically (Deshpande et al. 1997). In
vivo splicing rates for these introns are very slow
in the dark, but increase coordinately at least 6 to
10-fold within 15 min of light administration.
Evidence from inhibitor studies and analysis of
non-photosynthetic mutants indicates that the
light induction of psbA splicing requires electron
transport, but not ATP synthesis. The data
(Deshpande et al. 1997) also indicate that there is
no obligatory order for splicing of the psbA
introns, and that their rates of splicing in the light
are similar.
Recently, evidence was presented indicating
that the light stimulation of psbA splicing is
necessary for the maintenance of photosynthesis
(Lee and Herrin 2003). This was achieved by
making mutations in the Cr.psbA4 intron that
only partially inhibited splicing, and then examining the effects on production of the D1 protein,
which is encoded by the psbA gene, and photosynthetic growth. The most informative intron
mutation, a doubly substituted P4 helix mutant,
had a 45% reduction in Cr.psbA4 splicing, a 28%
reduction in synthesis of full-length D1, and an
18% reduction in photosynthetic growth rate.
Clearly, a 6- to 10-fold reduction in splicing of
these introns, which would be considered the
dark rate, would be expected to have an even
more negative effect on photosynthetic growth.
These results are important, since some chloroplast mRNAs appear to be in considerable excess
of what is required to maintain translation rates,
at least in short-term experiments with asynchronous cultures (Eberhard et al. 2002). However, this is clearly not the case for psbA mRNA
in longer-term growth studies.
Trans-acting genes for group I splicing
Some group I introns in fungal mitochondria
encode proteins that specifically promote splicing
of their host intron and other very closely related
introns. These proteins, originally called maturases, are usually coded in-frame with the upstream
exon, and contain a LAGLIDADG motif, which
is also found in intron-encoded endonucleases
(reviewed in Saldanha et al. 1993; Ho et al. 1997).
These maturases are thus bifunctional proteins. As
shown in Figure 1, three of the four psbA introns
(Cr.psbA2–Cr.psbA4) contain large ORFs, and for
Cr.psbA3, the ORF is in-frame with the upstream
exon; however, they do not have a recognizable
LAGLIDADG motif. In each case, most of the
ORF was deleted by using chloroplast transformation to replace the intron with an ORF-deleted
version. Analysis of psbA transcripts in these
strains revealed the absence of any effects on
splicing of the introns (Lee 2003; J. Lee, O.W.
Odom and D.L. Herrin, unpublished results).
Thus, based on these results, and the previous
analysis of the Cr.LSU intron ORF (Thompson
and Herrin 1991), the C. reinhardtii group I introns
do not appear to encode maturases that are
required for splicing of their host introns. At least
three of the introns, Cr.LSU, Cr.psbA2, and
Cr.psbA4 are mobile introns, and the ORFs thus
encode endonucleases specific for their exon junction sequences (Dürrenberger and Rochaix 1991;
Thompson et al. 1992; Dürrenberger et al. 1996;
Odom et al. 2001).
The apparent absence of maturase activity for
the intron ORFs suggests that such activities are
likely to be nuclear encoded. The nuclear ac20
mutant of C. reinhardtii is partially deficient in
splicing of the Cr.LSU intron (Herrin et al. 1990)
suggesting that this gene plays a role in splicing of
this intron. Subsequent work, however, showed
that the Cr.LSU splicing deficiency is not responsible for the slow-growth phenotype of ac20, and,
moreover, that it is also defective in removing the
internal transcribed spacers from the 23S rRNA
(Holloway and Herrin 1998). Thus, the ac20 gene
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product plays a more important role in processing
of the rRNA spacers than in splicing.
A successful approach for identifying nuclear
genes affecting chloroplast group I intron splicing
in C. reinhardtii was recently reported by Li et al.
(2002). These authors used suppressor genetics to
identify nuclear genes that promote splicing of
multiple group I introns. This approach was
selected because it was reasoned that mutants
blocked in splicing of these introns would be lethal
if the same proteins promoted splicing of the psbA
and Cr.LSU introns (chloroplast ribosomes are
essential for growth under all conditions (Harris et
al. 1994)). Despite the requirement of Cr.LSU
splicing for cell survival, this intron was chosen as
the primary target for suppressor analysis, because
of the extensive functional data on this intron
(Thompson and Herrin 1991; Kuo 1998; Kuo and
Herrin 2000a, b), and because it had already been
shown that the wild-type intron could be replaced
with mutants containing nucleotide substitutions
in the P4 helix (Holloway and Herrin 1998). The
splicing-deficient P4125A mutant, which exhibited
slow growth and light sensitivity, was used to
isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic
analysis of the 7151 and 7120 suppressors showed
that they define distinct nuclear genes, called css1
and css2 (chloroplast splicing suppressor), that are
also dominant in vegetative diploids (Li et al. 2002;
Lee 2003); the 71N1 suppressor may represent a
third gene linked to the 7151 suppressor. All three
of these suppressor strains also suppressed a
mutation in the P4 region of the Cr.psbA4 intron,
indicating that these genes play a role in splicing of
multiple group I introns. Cloning of these genes
should be feasible, and will allow their mechanism
of action to be studied in vitro and in vivo.
The draft sequence of the C. reinhardtii nuclear
genome was searched for genes related to the
mitochondrial group I splicing factors in fungi.
Two of the fungal factors are bifunctional tRNA
synthetases, tyrosyl and leucyl, whereas the others
(e.g., CBP2 and MRS1) are more idiosyncratic
genes (Dujardin and Herbert 1996; Lambowitz
et al. 1999). The mitochondrial tyrosyl tRNA
synthetase from Neurospora crassa, which is also
known as the CYT18 gene product, is the best
studied group I splicing factor. It can promote
splicing of most group I introns that do not have a
complex extension of the P5 region, including
Cr.LSU (Wallweber and Lambowitz, personal
communication); it has not been tested with the
Cr.psbA introns. Translated BLAST searches of
the Chlamydomonas genome sequence version 1.0
for these splicing factors revealed a candidate only
for the CYT18 gene (J. Lee and D.L. Herrin,
unpublished results). Unfortunately, not all of the
gene is present on the scaffold (2168), which terminates in the middle of the predicted protein. Of
great interest, however, is the fact that a 3-helix
motif, which is an idiosyncratic feature of CYT18
that is important for its splicing activity (Mohr
et al. 2001), is predicted for the N-terminus of this
protein. Work is underway to obtain the rest of the
protein sequence from a cDNA clone, and to
determine if this C. reinhardtii protein promotes
splicing of group I introns. It should also be noted
that this was the only tyrosyl-tRNA synthetase
gene in the C. reinhardtii genome of apparent
prokaryotic ancestry and with a predicted organellar targeting peptide, raising the possibility that
this protein is targeted to both mitochondria and
chloroplasts (D.L. Herrin, unpublished results).
Trans-acting factors required for splicing of the
psaA group II introns
Many of the early Photosystem I mutants of
C. reinhardtii turned out to be nuclear mutants
deficient in splicing of the trans-spliced psaA
introns (reviewed in Herrin et al. 1998). Three
different phenotypes were noted: (1) class C, deficient in splicing of the first intron; (2) class A,
deficient in splicing the second intron; and (3) class
B, deficient in splicing of both introns. Goldschmidt-Clermont et al. (1990) showed that these
mutants constitute 14 complementation groups:
five in class A, two in class B, and seven in class
C. One class A gene and one class C gene have been
cloned by complementation with cosmid libraries,
and the corresponding proteins identified. The
class A gene, Maa2 (or Raa2), encodes a protein
with sequence similarity to pseudouridine synthases, although, based on mutagenesis of putative
catalytic residues, this activity, if it has been retained by Raa2, is not required for its splicing
activity. Interestingly, this protein was found to be
associated with a chloroplast membrane fraction
that also contains other RNA-binding proteins
(Perron et al. 1999).
305
The class C gene, Raa3, encodes a large protein
of 1783 amino acids that has a small region of
similarity to pyridoxamine 50 -phosphate oxidases
(Rivier et al. 2001). Also, the N-terminal portion
of the protein is dispensable for its splicing promoting activity. The Raa3 protein was found in a
large soluble RNP complex containing the tscA
RNA (which contains part of the conserved core
of the intron) and the exon 1-containing transcript.
Apparently, Raa3 is necessary for the formation or
stabilization of this complex. As noted by the
authors, the splicing of this tripartite intron has
some interesting parallels with the splicing of
nuclear mRNA introns, which might even be
descendants of trans-spliced group II introns.
It should be noted that the Raa2 and Raa3
genes are unrelated to the crs or crs-associated
factor (caf1 and caf2) genes that promote splicing
of the cis-spliced group II introns in maize chloroplasts (Jenkins and Barkan 2001; Till et al. 2001;
Ostheimer et al. 2003). Also, BLAST searches
(E = 1 · 10)5) of the Chlamydomonas genome
sequence (version 1.0) with these genes did not
produce anything with the crs1, caf1, or caf2
genes, but a hit was obtained with the crs2 gene on
scaffold 435 (435.6). Protein targeting predictions
for the 435.6 ORF were ambiguous, however, with
respect to whether the protein might be chloroplast localized.
Nucleolytic processing of chloroplast transcripts
Most chloroplast transcripts are subject to nucleolytic processing at the 30 terminus, because transcription typically continues beyond the 30 end of
the gene. Inverted repeats occur downstream of
many chloroplast genes, and play an important
role in determining the 30 end of these mRNAs
(reviewed in Stern and Drager 1998). Many transcripts are also probably processed at the 50 end,
although this has not been as well studied. In
addition, internal nucleolytic processing occurs
within certain polycistronic transcripts, such as the
rRNA (reviewed in Harris et al. 1994), atpA and
psbB gene clusters (Stern and Drager 1998).
Proteins directly responsible for these maturation events have not been identified in C. reinhardtii.
However, a nuclear gene that is involved in 30 processing of multiple transcripts has been identified
genetically. The crp3 mutant was isolated as a sup-
pressor of a mutation in atpB mRNA that destabilized the transcript by removal of its 30 inverted
repeat; the crp3 mutation resulted in increased levels
of the discrete atpB transcript (Levy et al. 1997).
This recessive mutation targets the 30 UTR, and also
affects the 30 processing of other chloroplast transcripts (Levy et al. 1999). Thus, the crp3 gene may
encode an important component of the chloroplast
30 processing machinery.
Hahn et al. (1998) showed that a C. reinhardtii
mutant designated as a class B mutant in psaA
trans-splicing (HN31) is also defective in processing the tscA–chlN precursor transcript, a step that
may be necessary to generate the functional 30 end
of tscA RNA. Interestingly, however, this mutant
is also defective in splicing of the second psaA
intron, which does not require tscA, suggesting
that it plays a role in trans-splicing as well as
processing of the tscA–chlN transcript.
Another nuclear gene that should prove interesting to clone is the ac20 mutation, first described
more than 30 years ago (Boynton et al. 1970). This
mutation causes a reduction in levels of functional
chloroplast ribosomes, and a concomitant decrease in removal of the internal transcribed
spacers of the 23S rRNA precursor (Holloway and
Herrin 1998). Processing of the other parts of the
rRNA operon are unaffected in ac20.
Bruick and Mayfield (1998) presented evidence
that the processing of the 50 end of the psbA
mRNA is coupled to association of the pre-RNA
with ribosomes. 50 processing was inhibited in
trans-acting (nuclear) or cis-acting (50 UTR) mutants deficient in translation of psbA mRNA. The
nuclease involved could be associated with ribosomes; alternatively, ribosome binding could alter
the structure of the 50 end of the transcript making
it more susceptible to nucleolytic cleavage.
Several mutants of C. reinhardtii have been
isolated, and in some cases the corresponding
genes cloned, whose phenotypes involve a destabilized chloroplast mRNA. Some of these genes
may also play a role in processing of the transcript
in question; these genes are discussed below in the
context of RNA stability.
RNA stabilization
In the past, comparisons of chloroplast transcription rates with steady-state transcript levels
306
revealed that light or cell-cycle dependent changes
in RNA accumulation are determined by genespecific transcriptional as well as post-transcriptional processes affecting RNA half-lives in
C. reinhardtii (Leu et al. 1990; Salvador et al.
1993a). These effects appear to be superimposed
upon a general increase in transcription in the light
period, and enhanced RNA stability in the dark
(Nickelsen 1998). Furthermore, experiments in
which photosynthetic electron transport was
inhibited suggested that the RNA degradation
system – similar to the transcriptional and translational machinery – can be influenced by the
redox potential of the chloroplast stroma (Bruick
and Mayfield 1999; Pfannschmidt and Allen 1999;
Salvador and Klein 1999).
While these analyses were restricted to a few
selected genes, the recent completion of the chloroplast genome sequence from C. reinhardtii (Maul
et al. 2002), and establishment of plastome-wide
DNA microarrays, led to a more thorough investigation of chloroplast transcriptome fluctuations
(Lilly et al. 2002). Besides the previously recognized influence of different light conditions,
nutrient and UV-light stresses had substantial
effects on chloroplast RNA abundance. For
example, sulfur deprivation led to a 2- to 10-fold
decrease in transcriptional activity, which was
accompanied by a corresponding drop in mRNA
levels. However, removal of phosphate from the
growth medium resulted in a 2- to 3-fold increase
in RNA stability (Lilly et al. 2002).
However, the importance of gene-specific RNA
stabilization mechanisms is most significantly
illustrated by the isolation of nuclear mutants,
most of which exhibit a defect in the stability of a
single chloroplast RNA (Table 1). The affected
nuclear loci likely encode regulatory factors,
which, in concert with the basic machinery for
RNA turnover, determine transcript-specific halflives. Currently, the cloning of these genes from
C. reinhardtii, A. thaliana and Z. mays, represents
a key goal in this area, and which will allow us to
understand the molecular workings of these factors in more detail. To date, two RNA stability
factors from C. reinhardtii, Mbb1 (Vaistij et al.
2000b) and Mbd1 (Boudreau et al. 2000), have
been cloned, and a Mbb1-homologue, HCF107,
was independently identified in A. thaliana (Felder
et al. 2001).
Besides the characterization of the requisite
trans-acting factors, the localization of their target
sites is essential to elucidating the molecular
events leading to controlled RNA stabilization.
Whereas chloroplasts of A. thaliana and maize
cannot yet be efficiently transformed, chimeric
reporter genes or mutated versions of endogenous
genes can be easily introduced into the chloroplast
genome of C. reinhardtii via homologous recombination, thus allowing one to dissect the RNA
regions that influence transcript stability. Furthermore, with genetic crosses, the chloroplast
reporter genes can be combined with various
mutant nuclear backgrounds in order to deter-
Table 1. Nuclear mutations affecting chloroplast RNA stability
Mutantb
a
GE2.10
mbb1 (222E)a
6.2z5
mbd1 (nac2-26)
suPRB2A-1,-2,-3
MU11
MU37
mcd1 (F16)
mcd2
mda1 (ncc1)
thm24
tr72
crp3
a
b
Plastid target gene
Target RNA element
References
psbB
psbB
psbC
psbD
psbD
petA
petB
petD
petD
atpA
atpB
tscA
several
?
50 UTR
?
50 UTR
50 UTR
?
?
50 UTR
50 UTR
Coding region
?
?
30 UTR
Sieburth et al. (1991)
Vaistij et al. (2000a)
Sieburth et al. (1991)
Nickelsen et al. (1994)
Nickelsen (2000)
Gumpel et al. (1995)
Gumpel et al. (1995)
Drager et al. (1998)
Esposito et al. (2001)
Drapier et al. (2002)
Drapier et al. (1992)
Hahn et al. (1998)
Levy et al. (1999)
It has not been tested yet, whether GE2.10 and mbb1 are allelic.
Historically former mutant names are included in parentheses.
307
mine the target sites for the above-mentioned
nucleus-encoded factors.
In general, crucial cis-acting determinants for
RNA stability have been found in the untranslated
regions (UTRs) of chloroplast transcripts. One
exception is represented by the atpA mRNA whose
stability is controlled by the nuclear Mda1 locus.
By using chimeric gene constructs integrated into
the chloroplast genome, Drapier et al. (2002)
showed that the target for Mda1 function is the
coding region of the atpA mRNA. Additional
exceptional features of the mda1 mutant include
the facts that, in contrast to most other mutants
(Table 1), the atpA RNA still accumulated to a
level that allowed photoautotrophic growth, and
the mda1 mutation is dominant. In most other
cases studied so far, either the 50 or 30 UTRs of
chloroplast transcripts were shown to harbour the
essential RNA elements for stabilization.
30 UTR-mediated RNA stabilization and
polyadenylation
The first hints of the importance of 30 UTRs for
chloroplast RNA metabolism were derived from
in vitro assays with chloroplast extracts from vascular plants and exogenously-added labeled RNAs
(Stern and Gruissem 1987). In particular, inverted
repeat sequences, which can fold into so-called
stem-loop structures, attracted much attention,
since they resemble transcriptional terminators
from bacteria. However, both in vitro and in vivo
data indicate that these elements do not serve as
terminators, but instead function as RNA 30 -end
processing signals in both spinach and C. reinhardtii (Rott et al. 1996).
The most thoroughly analyzed 30 UTR from a
chloroplast transcript in C. reinhardtii is that of
atpB. This 30 UTR contains a stem-loop structure
which has a significant influence on both correct
30 processing and RNA stabilization, not unlike
the situation for the psaB mRNA (Stern et al.
1991; Lee et al. 1996). Interestingly, the stem-loop
containing 30 UTR of the spinach petD mRNA,
or a homopolymeric stretch of 18 consecutive G
residues, can functionally substitute for the atpB
30 region, suggesting that structurally different
RNA elements have the capability to protect
RNA regions from 30 to 50 exonucleolytic attack
in chloroplasts of C. reinhardtii (Drager et al.
1996). The mature 30 end of the atpB mRNA is
generated by a two step-process, which involves
an initial endonucleolytic cleavage 10-nt downstream of the stem-loop structure, followed by
exonucleolytic (30 to 50 ) trimming (Stern and
Kindle 1993). Besides its importance for atpB
gene expression, it has been shown that the 30
processing event has an additional interesting
molecular consequence. When the atpB IR, together with the endonucleolytic cleavage site, was
introduced into the 50 UTR of a chloroplast reporter transcript, the accumulation of reporter
mRNA was abolished. Apparently, sequences
located downstream of the atpB endonucleolytic
cleavage site are subject to rapid degradation,
probably by a wave of successive endonucleolytic
cuts (Hicks et al. 2002). This raises the possibility
that – at least in C. reinhardtii chloroplasts –
highly efficient degradation of RNAs downstream
of initial processing sites substitutes for an efficient transcription termination system. However,
it remains to be shown how the degradative
machinery differentiates between 50 ends which
are to be degraded and those which represent
mature termini of transcripts generated, for instance, by endonucleolytic processing of polycistronic messages.
In chloroplasts of vascular plants, and in
cyanobacteria, a polynucleotide phosphorylase
(PNPase) acts as both a 30 to 50 exonuclease, and
a poly(A) polymerase that adds multiple A residues to the 30 ends of chloroplast RNAs (Yehudai-Resheff et al. 2001; Rott et al. 2003). The
polyadenylation targets the RNAs for rapid
degradation by PNPase (Hayes et al. 1999;
Schuster et al. 1999). Also, in C. reinhardtii,
polyadenylation of all three major classes of
RNAs, i.e., mRNAs, tRNAs and rRNA, was
detected. In contrast to spinach, however, where
most polyadenylation sites coincide with endonucleolytic degradation sites within coding
regions (Lisitsky et al. 1997), in C. reinhardtii,
poly(A) tail formation was found to additionally
occur at precursor or mature 30 ends of transcripts (Komine et al. 2000). Recently, using an in
vivo approach, the role of such homopolymeric
tails within 30 UTRs has been analyzed in greater
detail. A poly(A) sequence was inserted downstream of either the atpB gene or a gfp reporter
together with a downstream tRNAGlu moiety,
which, after transcription and RNase P cleavage
308
upstream of the tRNA, leads to an exposed
poly(A) tail at the 30 RNA terminus. The chimeric transcripts were completely destabilized,
which also caused a photosynthesis-deficient
phenotype in the case of atpB (Komine et al.
2002). Unexpectedly, tails consisting mainly of U
residues also strongly reduced reporter RNA
accumulation, suggesting that the degradation
machinery is not specific for poly(A) regions.
Alternatively, multiple degradation pathways that
depend on homopolymeric RNA tails may exist
in the chloroplast of C. reinhardtii.
50 UTR-mediated RNA stabilization
The analysis of site-directed chloroplast mutants
and chloroplast reporter gene constructs has
revealed that 50 UTRs are also often important
determinants of RNA metabolism. The first indications for the role of chloroplast RNA 50 regions
were obtained for the rbcL gene (Salvador et al.
1993b). Precise mapping of RNA stability elements within the rbcL 50 UTR revealed a 10-nt
sequence required for RNA accumulation
(Anthonisen et al. 2001). Furthermore, a second
element located immediately upstream, appears to
be involved in a separate, light-dependent RNA
degradation mechanism (Singh et al. 2001). Interestingly, this region was shown to form a stable
stem-loop structure in vivo, suggesting that secondary structure elements similar to those in 30
UTRs represent critical determinants for RNA
longevity. Similar influences of RNA structures
have been implicated in the stabilization of atpB
and rps7 transcripts in C. reinhardtii (Fargo et al.
2000; Anthonisen et al. 2001). And in tobacco, 50
UTR-mediated regulation of rbcL mRNA turnover has been reported (Shiina et al. 1998).
Most strikingly, however, for most nucleusencoded RNA stability factors from C. reinhardtii
analyzed to date (Table 1), it has been demonstrated that their function is mediated – directly or
indirectly – via the 50 UTRs of the mRNAs they
regulate. These mRNAs include those of the psbD,
petD and psbB genes, which are unstable in the
respective nuclear mutants mbd1, mcd1 and mbb1
(Nickelsen et al. 1994; Drager et al. 1998; Vaistij
et al. 2000a). In contrast to the above mentioned
cases of mda1 and crp3, which act on the coding or
30 regions, respectively, in these three mutants,
RNA accumulation is completely abolished, as is
photoautotrophic growth. This clear phenotype
has facilitated the cloning of the Mbd1 and Mbb1
genes by complementation with a wild-type cosmid
library (Boudreau et al. 2000; Vaistij et al. 2000b).
Interestingly, the derived amino acid sequences
reveal that both proteins share a protein–protein
interaction motif called the TPR (tetratrico peptide) domain, which consists of multiple repeats of
a degenerate 34-amino acid motif (Blatch and
Lässle 1999). Also, Mbd1 and Mbb1 have been
found in distinct, stromal, high molecular weight
complexes of 600 and 300 kDa, respectively,
containing associated RNA molecules (Boudreau
et al. 2000; Vaistij et al. 2000b).
The precise molecular functions of these factors
from C. reinhardtii still have to be established, but
similar TPR motifs have recently been identified
in a Mbb1-homologue, HCF107, which is
involved in the processing/stabilization of psbH
transcripts in A. thaliana (Felder et al. 2001).
Furthermore, in vascular plants, a TPR-related,
so-called pentatricopeptide repeat motif was found
in two nucleus-encoded factors, Crp1 and
HCF152, which are required for the processing/
splicing of petA/petD or petB precursor transcripts, respectively (Fisk et al. 1999; Meierhoff et
al. 2003). For HCF152, direct binding to petB
precursor RNA was also demonstrated using
recombinant protein (Nakamura et al. 2003).
Searching the latest version of the C. reinhardtii
genomic sequence at JGI and the EST database
revealed no obvious homologues of Crp1 and
HCF152. Nevertheless, several genomic regions/
cDNAs were identified that show partial homology with PPR domains suggesting that – similar to
the situation in A. thaliana – multiple genes
encoding PPR proteins seem to be present within
the C. reinhardtii genome. Taken together, these
data suggest that the ancient TPR/PPR domain
fulfills an essential role in the organization of
functional units regulating RNA metabolism in
chloroplasts (Nickelsen 2003a). The cloning of
other nuclear loci that affect the stability of chloroplast RNAs (Table 1) will be necessary to
develop any general molecular principles for this
process.
A different genetic approach to identify loci
involved in RNA stability is based on the isolation
of mutant strains in which the primary mutations
in the nuclear genes or the chloroplast target
309
regions are phenotypically suppressed. Following
this strategy, a nucleus-encoded suppressor of the
mcd1 mutation that affects the stability of petD
mRNA (mcd2) was defined. In this suppressor,
petD mRNA accumulation is restored to 10% of
the wild-type level, and, in addition, petD translational efficiency is enhanced, suggestive of a dual
function for Mcd1 and Mcd2 (Esposito et al.
2001). Moreover, three unlinked nuclear suppressor loci have been shown to act on a mutation that
destabilized the psbD mRNA (Nickelsen 2000, see
below).
Besides genetic approaches to identify factors
regulating RNA stability, in vitro RNA-binding
studies have revealed that, in C. reinhardtii, several
proteins can interact directly with 50 UTRs
(Nickelsen 2003b). However, since 50 UTRs are
also generally the sites of translational regulation,
it is difficult to know which of the two processes
might be influenced by the RNA binding protein.
This is especially evident in view of the fact that
the processes of translation initiation and RNA
stabilization are linked at the molecular level (see
below).
The existence of nuclear gene products that
functionally interact with the 50 UTRs of specific
chloroplast mRNAs suggests that elements must
be present which are recognized by these factors,
and thus, are also required for RNA accumulation. Thorough mutagenesis studies have allowed
the mapping of such cis-acting sites in C. reinhardtii during the last several years. An 8-nt RNA
stability element was identified through linkerscanning mutagenesis at the very end of the petD 50
UTR. Dimethylsulfate modification experiments
revealed that this region forms a stem-loop structure that may interact with the nucleus-encoded,
Mcd1 factor (Drager et al. 1998; Higgs et al. 1999).
The psbD mRNA of C. reinhardtii is present in
two forms, a larger, low abundant precursor form
and a shorter, predominant form that is most
likely to be generated by a 50 processing event. By
applying site-directed mutagenesis of the 50 UTR,
two distinct elements required for stable accumulation of psbD mRNA have been mapped. In
contrast to petD, neither psbD element forms any
significant RNA secondary structure, as judged by
computer-assisted predictions and the analysis of
50 UTR mutants (Nickelsen et al. 1999). Similar to
petD though, a 12-nt element is located at the 50
terminal end of the psbD precursor transcript,
whereas the second one (PRB2, Figure 2) – 7 nt in
length – is located around position )30 relative to
the AUG start codon, and thus, is part also of the
mature psbD mRNA. Since the accumulation of
only the abundant, mature form of psbD mRNA is
Figure 2. Post-transcriptional regulation of chloroplast psbD gene expression in Chlamydomonas reinhardtii. The wild-type psbD 50
UTR sequence is shown with the elements required for RNA stabilization (boxed) or translation (grey). Below the sequence, names of
site-directed mutants and suppressor strains (in parentheses) are given, and the 50 processing site at position –47 (relative to the start
codon) that marks the 50 end of the abundant, mature psbD mRNA is indicated. See the text for further explanations.
310
affected in the nuclear mbd1 mutant, it has been
suggested that the PRB2 element represents the
Mbd1 recognition site (Nickelsen et al. 1999).
Three unlinked suppressor loci have been shown
to partially restore psbD mRNA accumulation in a
chloroplast PRB2 mutant and, hence, it was
speculated that these loci define different subunits
of the Mbd1 complex (Figure 2).
A similar situation has been found for the psbB
mRNA in C. reinhardtii, which also accumulates
as a longer, low-abundant precursor form and a
shorter 50 -processed mature form. In the nuclear
mutant, mbb1, only the mature RNA is absent
(Vaistij et al. 2000a), suggesting that Mbb1 and
Mbd1 are required for both RNA stabilization
and 50 processing.
Whilst the determinants for 50 UTR-mediated
RNA stabilization have been characterized to
some extent, relatively little is known about the
degradative machinery that attacks transcript 50
regions. The most important evidence was
obtained from the analysis of chloroplast transformants in which a stretch of 18 consecutive G
residues was inserted into various 50 UTRs of
plastid mRNAs. Similar to their protective function against PNPase-mediated 30 to 50 exonucleolytic degradation of 30 UTRs (see above), the 50
poly(G) tracts stabilized transcripts of the petD,
psbD and psbB genes in the respective nuclear
backgrounds of the mcd1, mbd1 and mbb1 mutants
(Drager et al. 1998; Nickelsen et al. 1999; Vaistij
et al. 2000a). This resembles the situation in yeast,
where poly(G) sequences have been shown to
impede the activity of 50 –30 exonucleases (Decker
and Parker 1993). Although a concerted action of
both endonucleolytic cuts and exonucleolytic
degradation – similar to the degradation of RNA
regions downstream of the atpB 30 processing site
(see above) – cannot be ruled out, the data
obtained for these three mRNAs clearly suggest
the presence of 50 –30 exonucleolytic activities in
chloroplasts of C. reinhardtii (Drager et al. 1999).
Whether a similar activity is also present in vascular plants has not yet been determined.
Relationship between RNA stability and translation
While the data summarized above clearly indicate
that levels of distinct chloroplast transcripts can be
determined post-transcriptionally, it has been
controversial as to whether or not a direct correlation exists between the level of a certain chloroplast mRNA and the level of the corresponding
protein. Several studies involving the analysis of
site-directed chloroplast mutants and the inhibition of translation with antibiotics give a complex
picture that does not reveal any general rule for
linkage of these two processes (for a review, see
Nickelsen 1998). In a recent, thorough investigation, it was found that amongst 8 chloroplast
mRNAs, including those of the psbA, psbD, petA,
petD, psaA, psaB, atpA and atpB genes, only the
atpB mRNA exhibited a direct correlation between
transcript abundance and translation rate,
following inhibition of chloroplast transcription
with rifampicin for 6 h (Eberhard et al. 2002). In
the other cases, no interrelationship was observed;
even when, for instance, petA or atpA mRNA
levels declined to 2% of the untreated control,
translation was almost not affected. As discussed
by the authors, this suggests that either the smaller
amounts of mRNA were intensively translated, or
that, under normal growth conditions, only a
minor fraction of these mRNAs is actively translated (Eberhard et al. 2002). The authors also
suggested that the rate-limiting determinant for
protein synthesis might be the availability of
nucleus-encoded factors that activate a subfraction
of a given mRNA species. In conclusion, these
data point to a minor role that RNA stabilization
events play in the regulation of chloroplast gene
expression, and that RNA metabolism and translation are not tightly linked. However, it should be
taken into account that experiments using inhibitors of chloroplast RNA synthesis allow the
observation of gene expression effects only over a
relatively short period of about 6 h. In contrast,
long-term growth effects could not be followed.
Moreover, as mentioned above with regard to
light-dependent psbA splicing, mutagenesis of the
Cr.psbA4 intron such that splicing was affected
only 45%, inhibited photosynthetic growth by
nearly 20%; thus, psbA mRNA is clearly not in
great excess. However, there does appear to be a
modest excess of this very abundant mRNA, at
least under the experimental conditions employed
(Lee and Herrin 2003).
Consequently, the importance of the abovementioned nucleus-encoded RNA stability factors
Mcd1, Mbd1 and Mbb1, needs to be assessed
311
further. As already mentioned, for all three it has
been shown that the insertion of a RNA-stabilizing poly(G) stretch into the respective petD, psbD,
and psbB 50 UTRs led to significant RNA accumulation even in the respective nuclear mutant
backgrounds. However, despite the presence of
almost wild-type mRNA levels, no corresponding
synthesis of subunit IV, D2, or CP47 occurred in
these strains. Since, in all cases, poly(G) insertion
had no effect on translational activity in a wildtype nulear background, these data indicate that in
addition to their role in RNA stabilization, the
nucleus-encoded factors are also required for
translation, and thus appear to represent molecular links between the processes of RNA longevity
and translation (Drager et al. 1998; Nickelsen et al.
1999; Vaistij et al. 2000a).
In the case of Mbd1 control of psbD gene
expression, this molecular relation has been analysed in greater detail, and a model is proposed that
is consistent with the available data (Figure 2).
Mbd1 is proposed to interact with the psbD 50
UTR at the PRB2 element once this region been
synthesized, and as a consequence, protects the
mRNA against exonucleolytic degradation from
the 50 end (Nickelsen et al. 1999). Subsequently,
Mbd1 appears to guide a protein of 40 kDa
(RBP40) to its cognate U-rich binding site, which
is located immediately downstream of PRB2, and
which has been shown to be required for psbD
translation (Ossenbühl and Nickelsen 2000). Once
this interaction has been established, ribosome
binding takes place and Mbd1 appears to be
released from the RNA, since it cannot be detected
in polysome fractions (Boudreau et al. 2000;
Nickelsen 2003b). Thus, Mbd1, together with
RBP40, appear to mediate the molecular linkage
between RNA stabilization and translation initiation, and, therefore, signify a close relationship
between these two processes.
Similar dual functions in both RNA metabolism and translation have been proposed for regulatory trans-acting factors from vascular plants,
e.g., Crp1 from maize or Hcf107 from A. thaliana
(Fisk et al. 1999; Felder et al. 2001), supporting the
idea of a general principle realized within chloroplasts. Taken together, currently available data
do not provide a general simple answer to the
question whether RNA stabilization and translation are tightly linked, and therefore future work
on this subject is needed.
Perspectives on future work
In regards to RNA splicing, the understanding of
light-promoted psbA splicing will require the
identification of the proteins that regulate this
process. It would also be useful to determine if the
precursors that accumulate in the dark are in an
unfolded or partially folded state. Although a
couple of the factors required for psaA transsplicing have been identified, additional ones will
have to be isolated before this process can be
understood in detail. It would also be satisfying to
verify that the tscA RNA interacts with the other
RNAs as suggested by the models. Development
of protein-dependent in vitro splicing systems
would also be of great help in studying lightdependent and trans-splicing.
Analysis of the signals that mediate intergenic
(or internal) processing of polycistronic transcripts
should be interesting, as would be the identification of the proteins that mediate nucleolytic processing in general.
With regard to RNA stability, although significant progress has been made in identifying
targets of trans-acting factors in the 50 UTRs, it
is not clear if these regions are responsible for
determining the half-lives of the mRNAs in wildtype cells. For example, we now know that 50
UTRs, 30 UTRs or coding regions can affect
RNA half-lives. Thus, a systematic determination
of which region is most important (i.e., limiting)
for a few mRNAs would be helpful in establishing the big picture. Also, additional
trans-acting factors need to be cloned, and it
needs to be determined if their expression is regulated. The identification of the nucleases involved
will be necessary to understand how trans-acting
factors protect mRNAs from them. Finally, the
connection between mRNA translation and
mRNA stability needs to be explored further, but,
to be most meaningful, it should be done in the
context of how much of the mRNA in question is
translated under a given set of conditions, and
how it affects growth or physiology.
The availability of the nuclear genome
sequence of Chlamydomonas should allow a systematic analysis of many of the gene products
targeted to chloroplasts and involved in RNA
processing and stability, using RNA interference
or anti-sense technology. The prediction of which
proteins are chloroplast targeted remains difficult
312
in some cases, however, so this approach will
probably miss some important proteins, but
should provide much useful information.
Acknowledgements
J. N. thanks U. Kück for basic support. J. N.’s
work is funded by the Deutsche Forschungsgemeinschaft (SFB480-B8). D. L. H. acknowledges financial support from the US Department
of Energy (DE-FG03-02ER15352), and the Robert A. Welch Foundation (F-1164).
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