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. 2012 Feb;63(3):1251-70.
doi: 10.1093/jxb/err347. Epub 2011 Nov 16.

Arabidopsis CSP41 proteins form multimeric complexes that bind and stabilize distinct plastid transcripts

Yafei Qi  1 , Ute Armbruster, Christian Schmitz-Linneweber, Etienne Delannoy, Andeol Falcon de Longevialle, Thilo Rühle, Ian Small, Peter Jahns, Dario Leister
Affiliations

Arabidopsis CSP41 proteins form multimeric complexes that bind and stabilize distinct plastid transcripts

Yafei Qi et al. J Exp Bot. 2012 Feb.

Abstract

The spinach CSP41 protein has been shown to bind and cleave chloroplast RNA in vitro. Arabidopsis thaliana, like other photosynthetic eukaryotes, encodes two copies of this protein. Several functions have been described for CSP41 proteins in Arabidopsis, including roles in chloroplast rRNA metabolism and transcription. CSP41a and CSP41b interact physically, but it is not clear whether they have distinct functions. It is shown here that CSP41b, but not CSP41a, is an essential and major component of a specific subset of RNA-binding complexes that form in the dark and disassemble in the light. RNA immunoprecipitation and hybridization to gene chips (RIP-chip) experiments indicated that CSP41 complexes can contain chloroplast mRNAs coding for photosynthetic proteins and rRNAs (16S and 23S), but no tRNAs or mRNAs for ribosomal proteins. Leaves of plants lacking CSP41b showed decreased steady-state levels of CSP41 target RNAs, as well as decreased plastid transcription and translation rates. Representative target RNAs were less stable when incubated with broken chloroplasts devoid of CSP41 complexes, indicating that CSP41 proteins can stabilize target RNAs. Therefore, it is proposed that (i) CSP41 complexes may serve to stabilize non-translated target mRNAs and precursor rRNAs during the night when the translational machinery is less active in a manner responsive to the redox state of the chloroplast, and (ii) that the defects in translation and transcription in CSP41 protein-less mutants are secondary effects of the decreased transcript stability.

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Figures

Fig. 1.
Fig. 1.
CSP41 insertion and overexpression lines: effects on CSP41 levels and plant growth. (A) The translated exons are numbered and shown as white boxes, with introns as black lines. Sites and orientations of T-DNA insertions are indicated. The csp41b-2 allele corresponds to the line SALK_021748 from the SALK T-DNA collection; csp41b-3 originates from the GABI-KAT collection and corresponds to GABI_452H11. The csp41a-3 and -4 alleles were identified in the T-DNA collection of C. Koncz (Max-Planck-Institute for Plant Breeding Research, Cologne, Germany) by PCR screening of lines originally designated as 91.154 and 61.888, respectively. (B) Western analysis of CSP41a in total protein extracts (40 μg) from young (7th–8th true) leaves of WT (Col-0), csp41a-3, csp41a-4, csp41b-2, and mature (3rd–4th true) leaves of csp41b-2. Decreasing amounts of WT extract were added to lanes marked 0.8× to 0.05× WT. The RbcL band visible after staining with Coomassie brilliant blue (C.B.B.) is shown as loading control. (C) Western analysis was performed on total protein extracts (40 μg) obtained from WT (Col-0) and csp41 mutant plants with CSP41a- and CSP41b-specific antibodies, and an antibody against Lhcb1 was used as a loading control. Note that csp41b-2 and csp41b-3 behaved identically (data not shown). (D) Phenotype of WT (Col-0) and csp41 mutant plants grown in the greenhouse. Here too, csp41b-2 and csp41b-3 behaved identically. (E) Growth kinetics of WT (Col-0) and csp41 mutant plants (n ≥10). Leaf area was measured during the period from 4 d to 28 d after germination (d.a.g.). Bars indicate standard deviations. Leaf area was always significantly (P < 0.01) lower for plants lacking CSP41b. (F) Development of WT (Col-0) and csp41 mutant plants (n ≥10). The number of true leaves was determined during the period from 4 to 28 d.a.g. Bars indicate standard deviations. Significant differences between WT/csp41a-4 and plants lacking CSP41b are indicated by two asterisks (P < 0.01). (G) Overexpression of CSP41a mRNA. Northern analysis of CSP41a mRNA from WT (Col-0), csp41a-4, csp41b-2, and 35S::CSP41a csp41b-2 (35S::CSP41a) plants. A CSP41a-specific probe and an ACTIN1-specific probe (as a control) were used. (H) Effects of CSP41a mRNA overexpression on accumulation of the CSP41a protein. Western analysis of WT (Col-0), csp41b-2, and 35S::CSP41a plants. Antibodies specific for CSP41a, CSP41b, and actin (as control) were used. (I) Phenotypes of WT (Col-0), csp41b-2, and 35S::CSP41a plants grown as in D.
Fig. 2.
Fig. 2.
Composition and synthesis of thylakoid proteins in greenhouse-grown csp41 mutants and the WT. (A) Western analysis was performed on total protein extracts (40 μg) obtained from WT (Col-0) and mutant plants. Decreasing amounts of WT extract were added to lanes marked 0.75× Col-0 and 0.5× Col-0. Protein complexes were visualized and quantified using antibodies specific for representative subunits. Actin was used as a loading control. As a further control for loading, a replicate gel was stained with Coomassie brillant blue (C.B.B). One representative of three independently prepared immunoblots is shown. (B) Pulse labelling of thylakoid membrane proteins in young (7th–8th true) leaves and mature (3rd–4th true) leaves of WT and csp41 mutants. After inhibition of cytosolic translation with cycloheximide, plastid protein synthesis was monitored by pulse-labelling with [35S]methionine for 20 min; thylakoid membrane proteins were isolated from WT and csp41 mutant leaves, fractionated by SDS–PAGE, and analysed by autoradiography. Prior to drying, the gel was stained with C.B.B. and the stained LHCII bands are indicated. The experiment was performed three times with similar results.
Fig. 3.
Fig. 3.
Characterization of complexes containing CSP41 proteins. (A) Total protein (TP) and chloroplast protein (Chl) extracts corresponding to 3 μg of Chl were subjected to SDS–PAGE, followed by immunoblot analysis using antibodies raised against CSP41 proteins. As controls, RbcL for chloroplast and actin for non-chloroplast proteins were used. As control, Ponceau Red stain (P.R.) of the membrane prior to antibody immunodecoration is shown. (B) Chloroplasts from WT plants were subfractionated into thylakoids (Thy), two envelope fractions (Env1 and Env2), and stroma (Str). Aliquots (40 μg) of proteins from each fraction were subjected to SDS–PAGE, followed by immunoblot analysis using antibodies raised against CSP41 proteins. As controls for the purity of the different fractions, antibodies recognizing Lhcb1, TIC110, and RbcL, which are located in thylakoids, envelope, and stroma, respectively, were used. (C) Stromal proteins from chloroplast samples corresponding to 30 μg of Chl were fractionated by BN-PAGE in the first dimension and by SDS–PAGE in the second. CSP41a and CSP41b were each detected using specific antibodies. The approximate molecular masses of the labelled protein complexes were estimated from the mobilities of other complexes with known molecular masses (Peltier et al., 2006). (D) Stromal proteins (70 μg) from WT (Col-0), csp41a-4 mutants, and a csp41b-2 line expressing CSP41b:CFP were cross-linked with EDC and fractionated by SDS–PAGE. Protein detection was carried out with specific antibodies as in A–C. Asterisks indicate unspecific bands detected by the CSP41b antibody.
Fig. 4.
Fig. 4.
CSP41 proteins are post-translationally modified. (A) Stromal proteins (500 μg) from WT (Col-0) and csp41b-3 plants were fractionated by IEF in the first dimension and by SDS–PAGE in the second. Proteins were transferred to PVDF membranes and stained with Coomassie brilliant blue (C.B.B.). (B) Sections of A containing CSP41 proteins stained with C.B.B. or immunodecorated with specific antibodies raised against CSP41a or CSP41b are shown. Arrows indicate the main CSP41a and CSP41b protein species. (C) The membranes from A were immunodecorated with antibodies against phosphorylated threonine residues; the detected signals were converted to red and merged with the image shown in A. Phosphorylated proteins are shown in purple, and highly phosphorylated proteins in red. (D) Sections of C containing CSP41 proteins. The asterisk marks glyceraldehyde-3-phosphate dehydrogenase B (Goulas et al., 2006). This protein, together with RbcL and RbcS, has been previously found to be phosphorylated (Lohrig et al., 2009; Reiland et al., 2009). Arrows indicate the main CSP41a and CSP41b protein species. (A–D) At least two independent experiments were performed with similar results.
Fig. 5.
Fig. 5.
CSP41-containing complexes interact with RNA. (A) Stromal proteins from dark-adapted plants corresponding to 160 μg of Chl were fractionated by BN-PAGE in the first dimension and by SDS–PAGE in the second. CSP41a and CSP41b were each detected using specific antibodies. As control, the Coomassie brilliant blue (C.B.B.) staining of the membrane prior to immunodecoration is shown. (B) Stromal proteins as in A, but treated with RNase. The asterisk indicates RNase A. (C) Stromal proteins from light-adapted WT and psad1-1 plants detected as in A. (A–C) The approximate molecular masses of the labelled protein complexes were estimated from the mobility of other complexes with known molecular masses (Peltier et al., 2006).
Fig. 6.
Fig. 6.
Identification of RNAs associated with CSP41b by RIP-chip analysis. (A) Differential enrichment ratios. The enrichment ratios (F635/F532) obtained from an assay involving CSP41b:CFP stroma were normalized with respect to a control assay using cp-eGFP stroma (both assays were performed in duplicate). The median normalized values for replicate spots from the cp-eGFP data were subtracted from the CSP41b data, log2 transformed, and plotted according to fragment number. Fragments are numbered according to their chromosomal positions. The data used to generate this figure are provided in Supplementary Table S3 at JXB online. Prominent peaks are labelled with gene names. (B) Differential pellet signals. The data from the experiments described in the upper panel were used to subtract normalized mean pellet signals (F635) obtained in cp-eGFP assays from those calculated for CSP41b:CFP assays. The corresponding data can also be found in Supplementary Table S4. (C) Slot-blot hybridization of RNAs that co-immunoprecipitate with CSP41b:CFP. One-tenth of the RNA recovered from each supernatant (sn) or half of the RNA recovered from each immunoprecipitation pellet (p) was applied to slot blots and hybridized with probes specific for rrn23, rrn16, psbA, rbcL, psaA, or psbC. (D) Slot-blot hybridization of RNAs that co-immunoprecipitate with CSP41a:eGFP and CSP41b:eGFP. One-tenth of the RNA recovered from each supernatant (sn) or half of the RNA recovered from each immunoprecipitation pellet (p) was applied to slot blots and hybridized with probes specific for rrn23, rrn16, psbA, rbcL, psaA, or psbC.
Fig. 7.
Fig. 7.
Lack of CSP41b affects the accumulation of chloroplast rRNAs and specific mRNAs. (A) Samples (20 μg) of total RNA from light-adapted WT (Col-0), csp41, and prpl11-1 mutant plants grown in the greenhouse (14 h/10 h light/dark regime at ∼180 μmol photons m−2 s−1) were size-fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and probed with cDNA fragments specific for the 5′ end of rrn23 and rrn16. Methylene blue (M.B.) staining of total leaf RNA served as loading control. The asterisk marks unprocessed rrn23. Quantification relative to WT can be found in Table 3. (B) Northern analyses of samples as in A with cDNA fragments specific for the coding regions of psbC, psbD, petD, psbE, rbcL, psaA/B, psbA, and ndhC. M.B. staining of total leaf RNA served as a loading control. (C) Run-on transcription in mature leaf chloroplasts isolated from WT and csp41b-2 plants. Duplicate nylon membranes were spotted with 1 μg or 0.5 μg of PCR products corresponding to the genes indicated at the top. Isolated chloroplasts were incubated in the presence of radiolabelled UTP. RNA from chloroplasts that had been pulse-labelled for 10 min was used to probe the membranes shown. The nuclear ACTIN1 gene was included as negative control. Quantification relative to rps18 can be found in Table 4. (D) Radiolabelled transcription products (TPs) from rrn23, psbC/D, and psbC were incubated with broken chloroplasts from WT and csp41b-2 mutants followed by re-isolation of RNA, separation on a denaturing 1.5% agarose gel, and transfer to nylon membranes. One representative of three independent experiments is shown. Note that in lane TP only one-tenth of the experimentally employed amount of transcription product was loaded. (A–D) Signals were analysed by autoradiography. (E) Working model for action of CSP41 protein complexes: in the light, newly synthesized rRNA precursors are rapidly incorporated into functional ribosomes, which in turn stabilize mRNAs during the translational process (arrows on ribosomes indicate translationally active ribosomes); CSP41 protein complexes do not bind to RNA. In the dark, CSP41 protein complexes associate with RNAs and protect them from nucleolytic cleavage. Untranslated RNA not stabilized by CSP41 protein complexes (i.e. in the csp41b and csp41ab double mutant) are degraded by RNases.
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