Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

Specific sequence elements in the59 untranslated regions of rbc Land atp B gene mRNAs stabilizetranscripts in the chloroplast ofChlamydomonas reinhardtii

INGER LILL ANTHONISEN, 1 MARIA L. SALVADOR, 2 and UWE KLEIN 1

1Department of Biology, University of Oslo, Blindern, 0316 Oslo, Norway2Department of Biochemistry and Molecular Biology, University of Valencia, 46100 Valencia, Spain

ABSTRACT

Using a series of point mutations in chimeric reporter gene constructs consisting of the 59 regions of the Chlamy-domonas chloroplast rbc L or atp B genes fused 59 to the coding sequence of the bacterial uid A (GUS) gene, RNA-stabilizing sequence elements were identified in vivo in the 59 untranslated regions (59 UTRs) of transcripts of thechloroplast genes rbc L and atp B in Chlamydomonas reinhardtii . In chimeric rbc L 59 UTR:GUS transcripts, replace-ment of single nucleotides in the 10-nt sequence 5 9-AUUUCCGGAC-39, extending from positions 138 to 147 relativeto the transcripts’ 5 9 terminus, shortened transcript longevity and led to a reduction in transcript abundance of morethan 95%. A similar mutational analysis of atp B 59 UTR:GUS transcripts showed that the 12-nt atp B 59 UTR sequence59-AUAAGCGUUAGU-3 9, extending from position 131 to position 142, is important for transcript stability and tran-script accumulation in the chloroplast of Chlamydomonas . We discuss how the 5 9 UTR sequence elements, which arepredicted to be part of RNA secondary structures, might function in RNA stabilization.

Keywords: 5 9 leader sequence; chloroplast gene transcripts; RNA longevity; RNA secondary structure

INTRODUCTION

Plastid genomes of higher plants and algae code for100–150 proteins, most of which have a function inphotosynthetic electron transport, ATP synthesis, ortranslation+ Their messenger RNAs have been found tobe relatively long-lived, having transcript-specific half-lives that range from a few hours to more than a day(Salvador et al+, 1993a)+ In addition to having transcript-specific longevities, chloroplast mRNAs have been foundto alter in stability during development (Klein & Mullet,1987), in response to external factors, for example,light (Simpson & Herrera-Estrella, 1990; Salvador et al+,1993a), and during endogenously controlled rhythms(Salvador et al+, 1993a; Hwang et al+, 1996)+

The components of the molecular machinery that de-grades mRNAs in chloroplasts are not known+ Homo-logs of the rne gene encoding RNase E, the principalendoribonuclease thought to be involved in mRNA deg-

radation in prokaryotes (Grunberg-Manago, 1999;Rau-hut & Klug, 1999), were found in three algal plastidgenomes—Porphyra purpurea (Reith & Munholland,1995), Nephroselmis olivacea (Turmel et al+, 1999),and Guillardia theta (Douglas & Penny, 1999)—(out of16 genomes sequenced to date), suggesting a roleof this enzyme in chloroplast mRNA breakdown+ InChlamydomonas, insertion of a poly(G) cassette,whichimpedes movement of exoribonucleases along RNAmolecules, into the 59 untranslated regions (UTRs) ofthe petD (Drager et al+, 1998, 1999) and psbB (Vaistijet al+, 2000) genes protected transcripts against rapiddegradation, suggesting the involvement of a 59-to-39exoribonuclease in mRNA decay in chloroplasts+ In ad-dition to factors acting at the 59 ends of mRNAs, factorsat the 39 ends of chloroplast transcripts appear to beimportant for mRNA stability+ Oligo(A) tails, supposedto be involved in bacterial mRNA decay (Sarkar, 1997;Grunberg-Manago, 1999), have been detected at the39 termini of chloroplast transcripts of Chlamydomonas(Komine et al+, 2000) and spinach (Lisitsky et al+, 1996),suggesting an oligo(A)-dependent pathway of mRNAdegradation in chloroplasts (Hayes et al+, 1999)+

Reprint requests to: Uwe Klein, Department of Biology, Universityof Oslo, Moltke Moes vei 32, 0316 Oslo, Norway; e-mail: uwe+klein@bio+uio+no+

RNA (2001), 7:1024–1033+ Cambridge University Press+ Printed in the USA+Copyright © 2001 RNA Society+

1024

Cold Spring Harbor Laboratory Press on July 24, 2016 - Published by rnajournal.cshlp.orgDownloaded from

http://rnajournal.cshlp.org/

http://www.cshlpress.com

Intramolecular determinants of mRNA longevity andthe molecular mechanisms responsible for control ofmRNA turnover in chloroplasts are largely unknown+ Anumber of studies show that the 59 UTRs of chloroplasttranscripts contain sequences that are crucial for mRNAstability (Salvador et al+, 1993b; Nickelsen et al+, 1994;Eibl et al+, 1999; Nickelsen, 1999; Singh et al+, 2001)+Analyses of nuclear mutants of Chlamydomonas thatfail to accumulate specific chloroplast transcripts iden-tified sites within the transcripts’ 59 UTRs that functionas cis-acting stability elements and probable targetsfor nucleus-encoded RNA-binding proteins (Nickelsenet al+, 1994; Higgs et al+, 1999; Nickelsen, 2000)+ Seg-ments that act as stability determinants were delin-eated within the 59 UTR sequences of transcripts of theChlamydomonas petD (Higgs et al+, 1999), psbD (Nick-elsen et al+, 1999), and psbB (Vaistij et al+, 2000) genesand within the 59 UTR of transcripts of the tobacco rbcLgene (Shiina et al+, 1998)+

Despite the evidence for a role of 59 untranslatedRNA sequences in determining mRNA longevities, cis-acting RNA-stabilizing elements in these regions havenot been defined or characterized in much detail todate+ Preliminary studies (I+L+ Anthonisen & U+ Klein,unpubl+), in which a number of random mutations wereintroduced into the 59 UTRs of transcripts of the Chlam-ydomonas chloroplast genes rbcL and atpB, led us tosuspect that the nucleotides around position 143 in therbcL 59 UTR and the nucleotides around position 137in the atpB 59 UTR are crucial for mRNA stability in theChlamydomonas chloroplast and, thus,might compriseelements that are involved in controlling transcript lon-gevity+ In this report, the results of mutational analysesare described that define two distinct transcript-specificsequence elements of about 10 nt in length required forhigh-level accumulation of transcripts in the chloroplastof C. reinhardtii+

RESULTS

Mutagenesis of rbc L and atp B 59UTR sequences

Transcripts of the genes rbcL and atpB, encoding thelarge subunit of ribulosebisphosphate carboxylase andthe b-subunit of the ATP synthase complex, are abun-dant in chloroplasts of C. reinhardtii (Salvador et al+,1993a)+Chloroplast transformants carrying chimeric re-porter genes in which 59 segments of the rbcL or atpBgene including the promoter and the first 64 nt of the 59UTRs were fused to the coding region of the bacterialuidA (GUS) gene, accumulate uidA transcripts to highlevels (Blowers et al+, 1990; Klein et al+, 1992)+ Half-lives of the chimeric 59 UTR:GUS transcripts have beendetermined to be around 5 h in cells kept in the dark (U+Klein, unpubl+), showing that the first 64 nt of the rbcLand atpB UTRs suffice to form RNA 59 ends that up-

hold stability of transcripts in the Chlamydomonas chlo-roplast+ The transcripts are not translatable becausethey lack the ribosome binding sites that are present inthe endogenous rbcL and atpB transcripts downstreamof the 164 positions+

Both 59 sequences are predicted to form RNAsecondary structures that fold the 59 terminus of thetranscripts into a double-stranded RNA conformation(Fig+ 1B)+ In vitro RNA alkylation with dimethylsulfate(DMS) coupled to reverse transcription, which de-tects unpaired adenines, cytosines, and uridines andallows us to assess the location of single- and double-stranded regions in RNA molecules (Moazed & Nol-ler, 1986), was used to verify the predicted secondarystructures of the 59 sequences of atpB and rbcL tran-scripts (Fig+ 1C)+ The methylation patterns of the 59regions of rbcL and atpB transcripts (Fig+ 1B,C) areconsistent with the RNA secondary structures pre-dicted for these sequences by the mfold program(Zuker et al+, 1999)+ Interestingly, the primer exten-sion results in conjunction with the DMS modificationpattern (Fig+ 1B,C) indicate the presence of atpB genetranscripts with two different 59 termini+ Primer exten-sion of the atpB 59 sequences resulted in two prom-inent cDNA fragments corresponding to the full lengthand a 25-nt shorter transcript (Fig+ 1C)+ The second-ary structures of the two 59 segments, as predictedby the mfold program (Zuker et al+, 1999), are shownin Figure 1B+ The DMS modification pattern does notsupport one of the two secondary structures shownin Figure 1B, but the pattern can be explained if bothstructures are present (Fig+ 1B,C)+ For example, theAUA sequence at the end of the left atpB 59 UTRstructure that should not be modified because it ispaired, occurs unpaired in the upper loop in the rightstructure+ The two paired but strongly modified ad-enines in the beginning of the right atpB UTR 59 struc-ture occur unpaired in the upper loop of the leftstructure+ Thus, the combined primer extension/DMSmodification data suggest that the primary atpB tran-script is processed at positions 125/126 (Fig+ 1A) toa shorter form that seems to be the more abundantof the two transcripts in our RNA samples+ The 59ends of both atpB transcripts are predicted to foldinto stem-loop conformations (Fig+ 1B)+

In a preliminary search for sequence elements inrbcL and atpB 59 UTRs that might act as RNA stabilitydeterminants, random point mutations were introducedinto positions 143, 149, 153, and 159 of the rbcL 59UTR and into positions 137,143,149, and 157 of theatpB 59 UTR (Fig+ 1A)+ In these analyses (I+L+ Antho-nisen & U+ Klein, unpubl+), it was noted that the nucle-otides in position 143 in the rbcL 59 UTR and in position137 in the atpB 59 UTR (Fig+ 1A) were crucial for sta-bility of transcripts of chimeric rbcL 59 end:GUS andatpB 59 end:GUS reporter genes+ To delineate pre-sumptive RNA-stabilizing sequence elements in these

Stability elements in chloroplast transcripts 1025




regions, the nucleotides adjacent to position 143 in therbcL 59 UTR and adjacent to position 137 in the atpB59 UTR were changed by random primer-mediated mu-

tagenesis (Fig+ 1A; Materials and Methods)+ rbcL andatpB 59 segments containing the point mutations de-picted in Figure 1A were fused to the coding region of

FIGURE 1. 59 UTR sequences (A), predicted RNA secondary structures (B), and dimethylsulfate modification (B, C) ofthe nucleotides of rbcL and atpB gene transcripts that were analyzed in this study for the presence of stability elements+ Thetotal lengths of the rbcL and atpB 59 UTRs is 92 and 340 nt, respectively (Dron et al+, 1982;Woessner et al+, 1986)+ A: The59 sequences fused in this study to the bacterial uidA gene+ Gray boxes in the sequences covering positions 138 to 147 inthe rbcL 59 UTR and positions 131 to 142 in the atpB 59 UTR mark the regions found in this work to be important fortranscript stability+ The individual single base changes introduced into the 59 UTR sequences are shown in the underlinedsequences below the gray boxed elements+ B: Secondary structures of the rbcL and atpB 59 sequences as predicted by themfold program (Zuker et al+, 1999)+ Boxed bases denote the location of the stability elements defined in this study (comparewith A)+ The two atpB 59 end structures shown correspond to the two transcripts probably present in the chloroplast asconcluded from the primer extension and DMS methylation data+ The atpB 59 UTR structure shown to the right lacks the25 terminal nucleotides of the primary transcript shown to the left+ Labels at the sequences indicate susceptibility to methyl-ation with DMS as estimated from the autoradiographs of C+ ✶: heavy methylation; d: medium methylation; C: weakmethylation+ Note that bases predicted by the mfold program to be unpaired are predominantly modified, and that modifi-cation of atpB 59 sequences is consistent with the presence of the two different structures shown (see text for explanation)+C: Methylation of rbcL and atpB 59 UTR sequences+Autoradiographs show the primer extension products of control and DMS-methylated 59 sequences of rbcL and atpB transcripts alongside sequencing ladders that serve as molecular weight markers(to the left of the samples loaded from left to right in the order A, T, G, C)+ Methylation blocks movement of the reverse tran-scriptase along ribonucleic acid strands+ The control lanes are loaded with samples that were not treated with DMS but wereotherwise processed like the DMS-modified samples+ Numbers to the right of the autoradiograms refer to the positions of the59 terminal nucleotides of the cDNAs, taking the 59 terminal nucleotide of the full-length cDNAs as 11+ Note that the methyl-ated nucleotides are located one position upstream of the depicted positions+ Exact sequence positions of the methylatedbases are marked in the predicted secondary structures of B+ (Figure continues on facing page+)

1026 I.L. Anthonisen et al.




the bacterial uidA (GUS) gene and stably introducedinto the chloroplast of C. reinhardtii by microprojectilebombardment (Boynton et al+, 1988)+ To assess theeffect of the mutations on transcript stability, the abun-dance of transcripts and rates of transcription of thechimeric rbcL 59 end:GUS and atpB 59 end:GUS re-porter genes were determined by RNA gel blot (north-ern) analyses and by in vivo pulse labeling assays,respectively (see Materials and Methods)+

Specific sequence elements in the rbc Land atp B 59 UTRs are important foraccumulation of transcripts in thechloroplast of Chlamydomonas

Levels of transcripts of chimeric rbcL 59 end:GUS genesare reduced markedly in Chlamydomonas chloroplasttransformants harboring chimeric genes with single pointmutations in the region extending from position 138 toposition 147 of the rbcL 59 UTR sequence (Fig+ 2)+Compared to GUS levels in a control transformant thatcarries the rbcL 59 UTR:GUS construct without muta-tions in the 59 UTR, reductions in mRNA levels rangedfrom 85% for changing the nucleotide in position 142[C r A] to almost 100% resulting from changes inseveral other positions (Fig+ 2)+ It should be noted thatin the latter cases, low levels of transcripts can be de-tected by prolonged exposure of the X-ray film to theRNA gel blot (see Fig+ 4)+ Nucleotide replacements insequences beyond positions 138 or 147 did not havea significant effect on levels of rbcL 59 UTR:GUS genetranscripts (Fig+ 2)+

In the 59 UTR of the atpB gene, positions 131 to 142are important for accumulation of transcripts of atpB 59

end:GUS genes in the chloroplast of Chlamydomonas.Nucleotide changes in this region resulted in decreasesin levels of GUS transcripts of 59% for a change inposition 133 to almost 100% due to a mutation in po-sition 138 (Fig+ 2)+ However, in general, mutations inthe sequence of the atpB 59 UTR did not result in re-ductions of GUS transcript levels as conspicuous asthose resulting from changes in the rbcL 59 UTR+ It ispossible that the presence of two species of atpB 59UTR:GUS transcripts in our samples—primary and pro-cessed (Fig+ 1B)—whose structures and/or stabilitiescould be affected differently by the introduced atpB 59end mutations, is responsible for the less pronouncedeffect on atpB 59 UTR:GUS transcript abundance+None-theless, apart from the 135 mutation that even causedtranscripts to accumulate to levels that were three timeshigher than levels of GUS transcripts in control cells(Fig+ 2), the effects are qualitatively comparable to theeffects on GUS transcript abundance in chloroplast

FIGURE 1. Continued.

FIGURE 2. Abundance of chimeric GUS transcripts in Chlamydo-monas chloroplast transformants harboring rbcL 59 end:GUS andatpB 59 end:GUS genes+ Total RNA was isolated from transformantsgrowing in 12-h light/12-h dark cycles at 11 h in the dark (rbcL:GUStransformants) or 1 h in the light (atpB:GUS transformants)+ RNAsamples were separated in a formaldehyde/1+3% agarose gel (Sam-brook et al+, 1989), blotted onto a nylon membrane (Zetaprobe, Bio-Rad), and hybridized overnight at 65 8C to the radiolabeled 1+9-kbGUS probe (see Materials and Methods)+ The washed membraneswere exposed to X-ray film with an intensifying screen for 10 h at280 8C+ Signals on the autoradiograms were quantified using Kodak1D image analysis software (Eastman Kodak Company, Rochester,New York)+ Numbers above the lanes denote the positions of theintroduced point mutations+ Numbers are relative to the 59 termini ofthe transcripts and refer to the sequence given in Figure 1+ C: control,that is, RNA isolated from a transformant carrying the 59 UTR se-quence without a mutation+





transformants harboring mutated rbcL 59 end:GUSconstructs+

Taken together, the results define sequences of about10–12 nt in length in the rbcL and atpB 59 UTRs thatare important for transcript accumulation in the chloro-plast of Chlamydomonas+

Mutations in the rbc L and atp B 59 UTRsaffect transcript stability

To assess to what extent the marked decrease in abun-dance of transcripts of chimeric rbcL 59 end:GUS andatpB 59 end:GUS genes resulting from changing singlenucleotides in the sequences of the 59 UTRs is causedby reduced synthesis or by reduced stability of the GUStranscripts, rates of transcription of rbcL 59 end:GUSand atpB 59 end:GUS genes were measured in vivo inseveral of the Chlamydomonas chloroplast transfor-mants (Fig+ 3)+ Transformants carrying rbcL 59 end:GUS genes with 59 UTR mutations in positions 140 or146 transcribed their chimeric GUS genes at the samerates as a control transformant carrying a rbcL 59 end:GUS gene with a 59 UTR mutation at position 148(Fig+ 3)+ The 148 transformant, which transcribes theGUS gene at the same rate as a previously analyzedcontrol transformant without a mutation in the rbcL 59UTR (Salvador et al+, 1993b), accumulated GUS tran-scripts to normal wild-type levels, whereas the 140and 146 transformants accumulated almost no GUStranscripts (Fig+ 2)+ Thus, it is concluded that changingthe nucleotides in positions 140 or 146 of the rbcL 59UTR does not affect transcription of the chimericGUS genes but renders the transcripts susceptibleto degradation+ The mutations in the other positionsof the rbcL 59 UTR sequence that lead to a loss ofGUS transcript accumulation comparable to the loss inthe 140 and 146 transformants (Fig+ 2) might alsoaffect the longevity of GUS transcripts rather than theirsynthesis+

The transformants carrying atpB 59 end:GUS geneswith mutations in the 131, 134, and 139 positions ofthe 59 UTR transcribe the chimeric GUS genes at ratescomparable to those found in the 130 control transfor-mant (Fig+ 3), which accumulates GUS transcripts tonormal wild-type levels (Fig+ 2)+ GUS gene transcrip-tion in the 130 transformant is as high as in previouslyanalyzed control transformants that carry a wild-typeatpB 59 UTR sequence (Klein et al+, 1992)+ In contrast,the mutation at position 140 resulted in a 30–40%reduction of GUS gene transcription when compared tothe rate of transcription of the endogenous atpB gene,and in a 60–70% reduction when compared to rates ofGUS gene transcription in the other atpB 59 end:GUStransformants analyzed+ Thus, while most of the ana-lyzed mutations in the atpB 59 UTR do not lower ratesof GUS gene transcription, and therefore identify nu-cleotides important for transcript stability, the effect of

the mutation in position 140 is indicative for the pres-ence of sequence elements in that region that affecttranscription+ The nucleotide at position 135 might alsobe part of such an element because its mutation re-sults in accumulation of GUS transcripts to levels thatare three times higher than transcript levels in the con-trol transformant (Fig+ 2)+A study of this transformant isunderway and will be presented elsewhere+

In summary, measurements of transcription ratesshow that all but one of the analyzed single point mu-tations introduced into the 59 UTR sequences of theChlamydomonas rbcL and atpB genes reduce tran-script abundance by lowering the stability of GUS re-porter gene transcripts in the chloroplast+

FIGURE 3. Rates of transcription of chimeric rbcL 59 end:GUS andatpB 59 end:GUS genes relative to the endogenous Chlamydomo-nas chloroplast genes rbcL and atpB+ Phosphate-starved cells wereallowed to incorporate [32P]-orthophosphate for 10 min and 20 min inthe dark+ Total RNA was isolated and hybridized for 72 h to probesspecific for the GUS, atpB, rbcL, and pUC18 sequences (see Ma-terials and Methods) spotted on a nylon membrane in a slot-blotapparatus (Hoefer Scientific Instruments, San Francisco, California)+Signals were visualized by exposure of the membranes to X-ray filmfor approximately 48 h and quantified using Kodak 1D image analy-sis software+ Numbers above the autoradiograms denote the posi-tions of mutated nucleotides in the rbcL or atpB 59 UTRs (see Fig+ 1)+Ratios given to the right of the autoradiograms were calculated rel-ative to the rates of phosphate incorporation into transcripts of theendogenous rbcL (upper panel) and atpB (lower panel) genes (set to1+00) that serve as internal standards+ Relative rates of transcriptionof rbcL 59 end:GUS genes are only 20 to 30% of the rate of tran-scription of the endogenous rbcL gene because the rbcL 59 endsfused to the GUS coding region lack a transcriptional enhancer thatis located in the coding region of the endogenous rbcL gene (Kleinet al+, 1994)+





Separate sequence elements in the rbc L 59UTR mediate longevity and light /darkstability of transcripts

It has been reported (Salvador et al+, 1993b) that tran-scripts of chimeric rbcL 59 end:GUS genes containingthe rbcL promoter and 59 UTR are rapidly degraded inChlamydomonas chloroplast transformants upon illu-mination of the cells+The light-induced decay was shownto be conferred to rbcL 59 UTR:GUS transcripts by therbcL 59 UTR sequence (Salvador et al+, 1993b)+ The invivo mutational analyses of the rbcL 59 UTR describedabove were done in cultures of Chlamydomonas grownin 12-h light/12-h dark cycles and cells for isolation oftotal RNA and for determination of rates of transcriptionwere collected at the end of the dark period (see Ma-terials and Methods)+ It seemed possible that the rbcL59 UTR sequence element (positions 138 to 147; Fig+ 1)found in this study to be required for stability of chime-ric rbcL 59 UTR:GUS transcripts is also involved inlight/dark regulation of transcript stability+ To test thisnotion, GUS transcript levels were determined in thedark and in the light in chloroplast transformants har-boring mutated rbcL 59 end:GUS genes (mutations atpositions 139, 140, 142, 145, and 146 in the rbcL 59UTR; Fig+ 4)+ GUS transcripts that were already desta-bilized by a mutation in the sequence element of therbcL 59 UTR and that accumulated in the dark to verylow levels in Chlamydomonas chloroplast transfor-mants (Fig+ 2), still showed the typical light/dark regu-lation of abundance (Fig+ 4)+ This suggests that thelight/dark-regulated mechanism of transcript (de-)

stabilization is distinct from the RNA decay mechanismfunctioning in turnover of rbcL chloroplast transcripts inthe dark, the former probably involving sequences inthe rbcL 59 UTR that are different from the elementdefined in this study (Singh et al+, 2001)+

DISCUSSION

The results presented in this study suggest that spe-cific 59 UTR sequences are crucial for stabilizing tran-scripts in the chloroplast of Chlamydomonas+ Fewanalyses have identified such sequences in chloroplasttranscripts (Fig+ 5), but none of the putative stabilizingelements has been characterized in much detail to date+The two sequences defined in this work are about 10 ntin length and reside in the 59 UTRs of rbcL and atpBgene transcripts in a region around 40 nt downstreamof the primary transcripts’ 59 termini+ They do not ap-pear to contain a consensus motif and are apparentlynot homologous to the other putative RNA-stabilizingelements delineated in chloroplast transcripts to date(Fig+ 5)+

The sequences are predicted to participate in theformation of RNA secondary structures (Fig+ 1B)+ InEscherichia coli, the presence of a hairpin structure atthe 59 ends of mRNAs has been found to be sufficientfor extending the half-life of transcripts (Emory et al+,1992;Arnold et al+, 1998), supposedly because bindingor activity of RNase E, the endoribonuclease thought todo the initial nucleolytic attack on bacterial mRNAs,requires a short stretch of free 59 terminal nucleotides(Bouvet & Belasco, 1992; Mackie, 2000)+ However, rel-atively stable E. coli transcripts, having half-lives of10–20 min, are still short-lived compared to chloroplasttranscripts that have half-lives of several hours+ Con-sidering the big difference in longevities of bacterialand chloroplast transcripts, and considering that onlyfew of the transcript-destabilizing mutations introducedin this work into the rbcL and atpB 59 UTRs are pre-dicted to destroy the presumptive original hairpin struc-tures at the transcripts’ 59 ends (as determined with themfold program; Zuker et al+, 1999), 59 end structuralfeatures alone are unlikely to be sufficient to prolonghalf-lives of transcripts to several hours in the Chlam-ydomonas chloroplast+

There is considerable experimental evidence for aninvolvement of transcript-specific nuclear-encoded RNA-binding proteins that protect the RNAs against nucle-olytic attack+ Analyses of several nuclear mutants ofChlamydomonas that do not accumulate specific chlo-roplast transcripts (Kuchka et al+, 1989; Sieburth et al+,1991; Drapier et al+, 1992; Nickelsen, 2000) showedthat nucleus-encoded protein factors are required fortranscript stability in the chloroplast+ Recently, the nu-clear Nac2 gene encoding a putative mRNA-stabilizingprotein that is required for accumulation of transcriptsof the Chlamydomonas chloroplast psbD gene has been

FIGURE 4. Light/dark regulation of GUS transcript levels in Chlam-ydomonas chloroplast transformants carrying rbcL 59 end:GUS genes+Transformants were grown in 12-h light/12-h dark cycles, total RNAwas isolated at time points 11 h dark and 1 h light, and processed asdescribed (legend to Fig+ 2; Materials and Methods)+ RNA gel blotswere first hybridized to the atpB probe as a control and, after strip-ping of the membrane, to the GUS probe+ The increase upon illumi-nation seen in abundance of transcripts of the endogenous atpBgene is due to increased transcription of the atpB gene in light andhas been reported previously (Salvador et al+, 1993a)+ The mem-brane was exposed to X-ray film for 4 h for detection of atpB genetranscripts and for 24 h for detection of chimeric rbcL 59 end:GUSgene transcripts+ Numbers above the lanes denote nucleotide re-placements in the rbcL 59 UTR sequence as depicted in Figures 1and 2+ D: dark (also marked by a filled bar); L: light (also marked byan open bar)+





isolated by genomic complementation (Boudreau et al+,2000)+ Putative cis-acting elements that are potentialtargets for RNA-stabilizing proteins have been local-ized in the 59 UTRs of chloroplast gene transcripts(Higgs et al+, 1999; Nickelsen et al+, 1999)+A number ofproteins were found in in vitro assays to bind to the59 UTRs of Chlamydomonas chloroplast transcripts(Hauser et al+, 1996) and, in spinach, to the 59 UTRs oftranscripts of genes that code for subunits of the ATPsynthase complex (Hotchkiss & Hollingsworth, 1999)+Although the identities and functions of these proteinshave not been examined, it is possible that some ofthem are nucleus-encoded factors involved in mRNAstabilization+

In view of these data, it is tempting to speculate thatthe sequence elements defined in this study representbinding sites for RNA-stabilizing proteins+ The protec-tive proteins could, for instance, function as stabilizersof the terminal hairpin structures that might hinder theinitial nucleolytic attack, for example, of an RNase E-typeendoribonuclease+ Consequently, mutations within thesequences would destroy the binding motif and/or theRNA secondary structures recognized by the stabiliz-ing protein resulting in less affinity and less frequentbinding of the protective proteins to the 59 UTR elements+

The finding that altering the sequence element in therbcL 59 UTR influences the longevity of transcripts inthe chloroplast of Chlamydomonas but does not affectthe light/dark regulation of transcript stability (Fig+ 4)points to a light/dark-regulated pathway of RNA break-down that differs from the general pathway of mRNAturnover in the chloroplast+ The light/dark-regulatedpathway has been shown to be linked to the redoxstate in the chloroplast (Salvador & Klein, 1999) andprobably involves chloroplast proteins that function asredox carriers+ The recent delineation in the rbcL 59UTR of cis-acting sequences at positions 121 to 141that play a role in light/dark regulation of rbcL transcriptstability (Singh et al+, 2001) should aid in identifying themolecular components and mechanisms involved incontrol of transcript longevities in chloroplasts+

MATERIALS AND METHODS

Algal cultures

C. reinhardtii, nonphotosynthetic mutant ac-uc-2–21 mt1 (atpBmutant CC-373) was obtained from the Chlamydomonas Ge-netics Center at Duke University, North Carolina, USA+ Themutant strain was grown in high salt (HS) medium (Sueoka,1960) containing 2+5 g potassium acetate per liter as de-scribed (Blowers et al+, 1990; Salvador et al+, 1993a)+ Trans-formants of the alga were grown as 100-mL cultures in liquidHS medium, either in 250-mL Erlenmeyer flasks or in glasstubes in a water bath at 32 8C+ Water bath cultures weremixed by bubbling with air supplemented with 2% CO2 (v/v)+Transformants grown for DNA isolation were cultured withcontinuous illumination (approximately 500 W/m2) whereastransformants to be used for isolation of RNA were grown ina 12-h light/12-h dark regime+

Chloroplast transformation

The chloroplast of the nonphotosynthetic atpB deletion mu-tant ac-uc-2–21 was transformed by microprojectile bombard-ment (Boynton et al+, 1988; Blowers et al+, 1989) using aPDS-1000/He Particle Delivery System (BioRAD)+ The vari-ous chimeric GUS genes cloned in the transformation vectorwere precipitated onto tungsten particles and bombarded intoagar-plated cells of mutant ac-uc-2–21+ Transformants wereselected on agar plates under photoautotrophic growth con-ditions as described (Blowers et al+, 1989)+ Transformantswere screened for the presence of the GUS constructs byDNA slot-blot (Southern) analysis and, when necessary, re-plated on HS agar plates to select for homoplasmic cell lines+

Polymerase chain reactions

Taq DNA polymerase (Promega) and Dynazyme (Finnzymes)were used in PCR amplifications of the 59 regions of theChlamydomonas chloroplast atpB and rbcL genes, respec-tively+Amplification was done in 30 cycles, each consisting ofa 1-min denaturation step at 94 8C, a 1-min annealing step at42 8C, and a 2-min elongation step at 72 8C+ After 30 cycles,the reactions were completed by keeping the samples for10 min at 72 8C+ The template for amplification of atpB 59

FIGURE 5. Sequences in 59 UTRs of transcripts of Chlamydomonas chloroplast genes that have been identified to date tobe important for mRNA stability+





sequences was plasmid pCrc32 containing ;220 bp of the 59region of the Chlamydomonas chloroplast atpB gene (Blow-ers et al+, 1993)+ The plasmid used as template for amplifi-cation of rbcL 59 UTR sequences contained a blunt-ended227 bp DNA fragment from the 59 end of the Chlamydomonaschloroplast rbcL gene (extending from position 270 to posi-tion 1157 relative to the site of transcription initiation) clonedinto the the EcoRV site of pBluescript SK1 (Stratagene)+

In vivo determination of transcription rates

Relative rates of transcription of the chimeric rbcL:GUS andatpB:GUS genes, and of the endogenous chloroplast genesrbcL and atpB were measured in vivo by labeling RNA with[32P]-orthophosphate as described (Blowers et al+, 1990)+

Construction of chimeric rbc L 59 end:GUSand atp B 59 end:GUS genes andDNA probes

Common molecular techniques for DNA manipulations wereused as described (Sambrook et al+, 1989)+ The transforma-tion vector pCrc32 (Blowers et al+, 1993) was the startingplasmid for all chimeric GUS constructs used in this work+The vector contains the complete Chlamydomonas chloro-plast atpB gene and a chimeric GUS gene consisting of thepromoter and 59 UTR sequences of the atpB gene fused tothe coding region of the E. coli uidA (GUS) gene and termi-nated by the 39 end of the Chlamydomonas chloroplast psaBgene+ For insertion of DNA fragments in front of the GUSsequence, the atpB promoter and 59 UTR sequences wereremoved by digestion of pCrc32 with XhoI/SmaI, which cut atunique restriction sites upstream of the GUS coding region+

The DNA segment between positions 270 and 163 rela-tive to the start of rbcL gene transcription containing the rbcLgene promoter and 63 bp of rbcL 59 UTR sequence wasamplified by PCR using the M13 universal primer 59-GTAAAACGACGGCCAGT-39 as 59 primer and the 39-mer 59-TTATCGATCCTAAAATAATCTGTCCGGAAATATAATTTA-39as 39 primer+ The 39 primer, which is complementary to po-sitions 130 to 168 of the rbcL 59 UTR sequence, was syn-thesized with a ClaI restriction site [ATCGAT] at its 59 endthat was used in subcloning the PCR-amplified DNA frag-ment into pBluescript SK1+ A series of variants of the 39primer containing single nucleotide changes in the positionsshown in Figure 1 were used to amplify rbcL 59 DNA frag-ments with mutated rbcL 59 UTR sequences+ Amplified rbcL59 fragments were digested with XhoI, which cuts at the 59end of the amplified rbcL gene region, and ClaI, which cuts atthe restriction site introduced by the 39 primer at position 163of the rbcL UTR sequence, and cloned into XhoI/ClaI-digestedpBluescript SK1+ The rbcL 59 DNA fragments were releasedfrom pBluescript SK1 by digestion with XhoI and SmaI andcloned into the XhoI/SmaI-digested transformation vectorpCrc32+

DNA fragments from the 59 end of the atpB gene contain-ing the sequence between positions 2120 to 162 relative tothe start site of atpB gene transcription were amplified byusing the 21-mer 59-GTGCAGTGCCCCCTCGAGGTC-39 as59 primer and the 45-mer 59-GAATTTAAATATAAAAAGTATTATTCACTAACGCTTATTTTTTAG-39 as 39 primer+ The lat-

ter is complementary to positions 124 to 168 of the atpB 59UTR sequence and contains a DraI restriction site [TTTAAA]at its 59 end that was used in subcloning of the PCR productsinto pBluescript SK1+ Variants of the 39 primer containing thenucleotide changes shown in Figure 1 were used to amplifyatpB 59 DNA segments with mutated 59 UTR sequences+Amplification products were digested with XhoI, which cuts atthe 59 end of the amplified atpB 59 fragment, and DraI, whichcuts in the atpB 59 UTR at position 162, and cloned intoXhoI/EcoRV-digested pBluescript SK1+ The fragments werereleased by digestion with XhoI and SmaI and cloned intoXhoI/SmaI-digested transformation vector pCrc32+ The se-quences of all cloned fragments were verified by Sanger di-deoxynucleotide sequencing+

It was not checked whether the transcripts of the chime-ric constructs are translated into the GUS (b-glucuronidase)protein+ For transcripts of the atpB 59 end:GUS constructs,translation is unlikely because they lack about 280 bp ofthe full-length atpB 59 UTR+ Translation of transcripts of therbcL 59 end:GUS constructs is doubtful because they lackabout 30 bp of the rbcL 59 UTR including the putativeribosome-binding (Shine–Dalgarno) motif at positions 171to 174 (Dron et al+, 1982)+

The ;1+9-kb BamHI-SacI restriction fragment from plas-mid pBI221 (Clontech, California, USA) containing the entirecoding region of the bacterial uidA (GUS) gene was used asGUS gene probe+ The ;890-bp HindIII restriction fragmentof plasmid pCrcrbcL (Blowers et al+, 1990) containing an in-ternal portion of the Chlamydomonas chloroplast rbcL genewas used as rbcL gene probe+ The atpB probe was an;700-bp HpaI/EcoRV restriction fragment from the proteincoding sequence of the Chlamydomonas chloroplast atpBgene released from the vector pCrcatpB (Blowers et al+, 1990)by cutting with the restriction endonuclease EcoRI+

Southern and northern blots

Genomic DNA and total RNA were isolated from C. rein-hardtii as described (Dellaporta et al+, 1983; Merchant &Bogorad, 1986; Salvador et al+, 1993a)+ DNA was isolatedfrom cultures growing under continuous illumination+ RNAwas isolated from cultures growing in a 12-h light/12-h darkregime+ When not specified differently, time points for iso-lation of total RNA were 11 h in the dark or 1 h in the lightfor transformants carrying rbcL:GUS genes and atpB:GUSgenes, respectively+ The presence of the chimeric GUS geneconstructs in Chlamydomonas chloroplast transformants wasanalyzed by DNA slot-blot hybridizations using the randomprimer-labeled (Feinberg & Vogelstein, 1983) GUS se-quence as probe+ The relative abundance of GUS tran-scripts in Chlamydomonas chloroplast transformants wasdetermined by RNA gel (northern) blots+ DNA and RNAblots were hybridized to radiolabeled probes and processedfor autoradiography as described (Church & Gilbert, 1984;Blowers et al+, 1990)+

Chemical modification of RNAwith dimethylsulfate

Experimental assessment of RNA secondary structures wasdone by base methylation essentially as described (Moazed





& Noller, 1986; Chen et al+, 1991)+ Briefly, 20–50 mg of totalChlamydomonas RNA were incubated in 80 mM MOPS,pH 7+2, 0+3 M KCl, 50 mM MgCl2, with 0+3% (v/v) dimethyl-sulfate (DMS) for 10 min at 30 8C (in a final incubation volumeof 100 mL)+ The modified RNA was ethanol precipitated andreverse transcribed with Superscript IITM (Gibco BRL) follow-ing the protocol that came with the enzyme+ The [32P]-end-labeled oligonucleotides 59-TGCCTAGTATGTAAACATTGTGGC-39 and 59-ACCTGCTTTAGTTTCTGTTTGTGGAACCAT-39, both complementary to sequences about 130 ntdownstream of the presumed 59 ends of the atpB and rbcLgene transcripts, respectively, were used as primers for re-verse transcription of the atpB and rbcL transcripts’ 59 re-gions+ Samples were extracted once with phenol/chloroformand once with chloroform, ethanol precipitated, and dissolvedin 4 mL sterile water 1 2 mL gel loading buffer (95% formam-ide, 20 mM EDTA, 0+05% bromophenol blue, 0+05% xylenecyanol FF)+ Two microliters of each sample were loaded on8% polyacrylamide sequencing gels and cDNAs were sepa-rated at 1,700 V alongside control samples containing cDNAsderived from unmodified RNA and alongside sequencing lad-ders that served as molecular weight marker+

ACKNOWLEDGMENTS

Parts of the work were supported by grants from the Norwe-gian Research Council (Grant No+ 100946/410 to U+K+) andthe Dirección General de Investigacion Cientifica y Technica(Grant No+ PB 98–1445 to M+L+S+)+

Received July 26, 2000; returned for revisionAugust 15, 2000; revised manuscript receivedApril 27, 2001

REFERENCES

Arnold TE, Yu J, Belasco JG+ 1998+ mRNA stabilization by the ompA59 untranslated region: Two protective elements hinder distinctpathways for mRNA degradation+ RNA 4:319–330+

Blowers AD, Bogorad L, Shark KB, Sandford JC+ 1989+ Studies onChlamydomonas chloroplast transformation: Foreign DNA can bestably maintained in the chromosome+ Plant Cell 1:123–132+

Blowers AD, Ellmore GS, Klein U, Bogorad L+ 1990+ Transcriptionalanalysis of endogenous and foreign genes in chloroplast trans-formants of Chlamydomonas+ Plant Cell 2:1059–1070+

Blowers AD, Klein U, Ellmore GS, Bogorad L+ 1993+ Functionalin vivo analyses of the 39 flanking sequences of the Chlamy-domonas chloroplast rbcL and psaB genes+ Mol Gen Genet238:339–349+

Boudreau E, Nickelsen J, Lemaire SD, Ossenbuhl F, Rochaix JD+2000+ The Nac2 gene of Chlamydomonas encodes a chloroplastTPR-like protein involved in psbD mRNA stability+ EMBO J 19:3366–3376+

Bouvet P, Belasco JG+ 1992+ Control of RNase E-mediated RNAdegradation by 59-terminal base pairing in E. coli+ Nature 360:488–491+

Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, JonesAR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB,Sanford JC+ 1988+ Chloroplast transformation in Chlamydomonaswith high velocity microprojectiles+ Science 240:1534–1538+

Chen L-H, Emory SA, Bricker AL, Bouvet P, Belasco JG+ 1991+ Struc-ture and function of a bacterial mRNA stabilizer:Analysis of the 59untranslated region of ompA mRNA+ J Bacteriol 173:4578–4586+

Church G,Gilbert W+ 1984+Genomic sequencing+ Proc Natl Acad SciUSA 81:1991–1995+

Dellaporta SL,Wood J, Hicks JB+ 1983+A plant DNA minipreparation:Version II+ Plant Mol Biol Rep 1:19–21+

Douglas SE, Penny SL+ 1999+ The plastid genome of the cryptophytealga, Guillardia theta: Complete sequence and conserved syn-teny groups confirm its common ancestry with red algae+ J MolEvol 48:236–244+

Drager RG, Girard-Bascou J, Choquet Y, Kindle KL, Stern DB+ 1998+In vivo evidence for 59 r 39 exoribonuclease degradation of anunstable chloroplast mRNA+ Plant J 13:85–96+

Drager RG, Higgs DC, Kindle KL, Stern DB+ 1999+ 59 to 39 exoribo-nucleolytic activity is a normal component of chloroplast mRNAdecay pathways+ Plant J 19:521–531+

Drapier D, Girard-Bascou J,Wollman FA+ 1992+ Evidence for nuclearcontrol of the expression of the atpA and atpB chloroplast genesin Chlamydomonas+ Plant Cell 4:283–295+

Dron M, Rahire M, Rochaix J-D+ 1982+ Sequence of the chloroplastDNA region of Chlamydomonas reinhardtii containing the gene ofthe large subunit of ribulose bisphosphate carboxylase and partsof its flanking genes+ J Mol Biol 162:775–793+

Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop HU+ 1999+ In vivoanalysis of plastid psbA, rbcL, and rpl32 UTR elements by chlo-roplast transformation: Tobacco plastid gene expression is con-trolled by modulation of transcript levels and translation efficiency+Plant J 19:333–345+

Emory SA, Bouvet P, Belasco JG+ 1992+ A 59-terminal stem-loopstructure can stabilize mRNA in Escherichia coli+ Genes & Dev6:135–148+

Feinberg AP, Vogelstein B+ 1983+ A technique for radiolabeling DNArestriction endonuclease fragments to high specific activity+ AnalBiochem 132:6–13+

Grunberg-Manago M+ 1999+ Messenger RNA stability and its role incontrol of gene expression in bacteria and phages+ Annu RevGenet 33:193–227+

Hauser CR, Gillham NW, Boynton JE+ 1996+ Translational regulationof chloroplast genes+ Proteins binding to the 5-untranslated re-gion of chloroplast mRNAs in Chlamydomonas reinhardtii+ J BiolChem 271:1486–1497+

Hayes R, Kudla J, Gruissem W+ 1999+ Degrading chloroplast mRNA:The role of polyadenylation+ Trends Biochem Sci 24:199–202+

Higgs DC, Shapiro RS, Kindle KL, Stern DB+ 1999+ Small cis-actingsequences that specify secondary structures in a chloroplastmRNA are essential for RNA stability and translation+ Mol CellBiol 19:8479–8491+

Hotchkiss TL, Hollingsworth MJ+ 1999+ATP synthase 59 untranslatedregions are specifically bound by chloroplast polypeptides+ CurrGenet 35:512–520+

Hwang S, Kawazoe R, Herrin DL+ 1996+ Transcription of tufA andother chloroplast-encoded genes is controlled by a circadian clockin Chlamydomonas+ Proc Natl Acad Sci USA 93:996–1000+

Klein RR, Mullet JE+ 1987+ Control of gene expression during higherplant chloroplast biogenesis+ Protein synthesis and transcript lev-els of psbA, psaA-psaB, and rbcL in dark-grown and illuminatedbarley seedlings+ J Biol Chem 262:4341–4348+

Klein U, De Camp JD, Bogorad L+ 1992+ Two types of chloroplastgene promoters in Chlamydomonas reinhardtii+ Proc Natl AcadSci USA 89:3453–3457+

Klein U, Salvador ML, Bogorad L+ 1994+ Activity of the Chlamydo-monas rbcL gene promoter is enhanced by a remote sequenceelement+ Proc Natl Acad Sci USA 91:10819–10823+

Komine Y, Kwong L, Anguera MC, Schuster G, Stern DB+ 2000+Polyadenylation of three classes of chloroplast RNA in Chlamy-domonas reinhardtii. RNA 6:598–607+

Kuchka M, Goldschmidt-Clermont M, Van Dillewijn J, Rochaix J-D+1989+ Mutation at the Chlamydomonas nuclear NAC2 locus spe-cifically affects stability of the chloroplast psbD transcript encod-ing polypeptide D2 of PSII+ Cell 58:869–876+

Lisitsky I, Klaff P, Schuster G+ 1996+Addition of destabilizing poly(A)-rich sequences to endonuclease cleavage sites during the deg-radation of chloroplast mRNA+ Proc Natl Acad Sci USA 93:13398–13403+

Mackie GA+ 2000+ Stabilization of circular rpsT mRNA demonstratesthe 59-end dependence of RNase E action in vivo+ J Biol Chem275:25069–25072+

Merchant S, Bogorad L+ 1986+ Regulation by copper of the expres-sion of plastocyanin and cytochrome c552 in Chlamydomonasreinhardtii+ Mol Cell Biol 6:462–469+

Moazed D, Noller HF+ 1986+ Transfer RNA shields specific nucleo-





tides in 16S ribosomal RNA from attack by chemical probes+ Cell47:985–994+

Nickelsen J+ 1999+ Transcripts containing the 59 untranslated regionsof the plastid genes psbA and psbB from higher plants are un-stable in Chlamydomonas reinhardtii chloroplasts+Mol Gen Genet262:768–771+

Nickelsen J+ 2000+ Mutations at three different nuclear loci of Chlam-ydomonas suppress a defect in chloroplast psbD mRNA accu-mulation+ Curr Genet 37:136–142+

Nickelsen J, Fleischmann M, Boudreau E, Rahire M, Rochaix J-D+1999+ Identification of cis-acting RNA leader elements requiredfor chloroplast psbD gene expression in Chlamydomonas. PlantCell 11:957–970+

Nickelsen J, van Dillewijn J, Rahire M, Rochaix J-D+ 1994+ Determi-nants for stability of the chloroplast psbD RNA are located withinits short leader region in Chlamydomonas reinhardtii+ EMBO J13:3182–3191+

Rauhut R, Klug G+ 1999+mRNA degradation in bacteria+ FEMS Micro-biol Rev 23:353–370+

Reith ME, Munholland J+ 1995+ Complete nucleotide sequence ofthe Porphyra purpurea chloroplast genome+ Plant Mol Biol Rep13:333–335+

Salvador ML, Klein U+ 1999+ The redox state regulates RNA degra-dation in the chloroplast of Chlamydomonas reinhardtii+Plant Phys-iol 121:1367–1374+

Salvador ML, Klein U, Bogorad L+ 1993a+ Light-regulated and en-dogenous fluctuations of chloroplast transcript levels in Chlamy-domonas+Regulation by transcription and RNA degradation+ PlantJ 3:213–219+

Salvador ML, Klein U, Bogorad L+ 1993b+ 59 sequences are importantpositive and negative determinants of the longevity of Chlamy-domonas chloroplast gene transcripts+ Proc Natl Acad Sci USA90:1556–1560+

Sambrook J, Fritsch EF, Maniatis T+ 1989+ Molecular cloning: A lab-

oratory manual. Cold Spring Harbor, New York: Cold Spring Har-bor Laboratory Press+

Sarkar N+ 1997+ Polyadenylation of mRNA in prokaryotes+ Annu RevBiochem 66:173–197+

Shiina T, Allison L, Maliga P+ 1998+ rbcL transcript levels in to-bacco plastids are independent of light: Reduced dark transcrip-tion rate is compensated by increased mRNA stability+ PlantCell 10:1713–1722+

Sieburth LE, Berry-Lowe S, Schmidt GW+ 1991+ Chloroplast RNAstability in Chlamydomonas:Rapid degradation of psbB and psbCtranscripts in two nuclear mutants+ Plant Cell 3:175–189+

Simpson J, Herrera-Estrella L+ 1990+ Light-regulated gene expres-sion+ Crit Rev Plant Sci 9:95–109+

Singh M, Boutanaev A, Zucchi P, Bogorad L+ 2001+ Gene elementsthat affect the longevity of rbcL sequence-containing transcriptsin Chlamydomonas reinhardtii chloroplasts+ Proc Natl Acad SciUSA 98:2289–2294+

Sueoka N+ 1960+Mitotic replication of deoxyribonucleic acid in Chlam-ydomonas reinhardi. Proc Natl Acad Sci USA 46:83–91+

Turmel M, Otis C, Lemieux C+ 1999+ The complete chloroplast DNAsequence of the green alga Nephroselmis olivacea: Insights intothe architecture of ancestral chloroplast genomes+ Proc Natl AcadSci USA 96:10248–10253+

Vaistij FE,Goldschmidt-Clermont M,Wostrikoff K, Rochaix J-D+ 2000+Stability determinants in the chloroplast psbB/T/H mRNAs ofChlamydomonas reinhardtii+ Plant J 21:469–482+

Woessner JP, Gillham NW, Boynton JE+ 1986+ The sequence of thechloroplast atpB gene and its flanking regions in Chlamydomo-nas reinhardtii+ Gene 44:17–28+

Zuker M, Mathews DH, Turner DH+ 1999+ Algorithms and thermo-dynamics for RNA secondary structure prediction:A practical guide+In: Barciszewski J, Clark BFC, eds+ RNA biochemistry and bio-technology+ NATO ASI Series, Kluwer Academic Publishers+ pp11–43+





2001 7: 1024-1033 RNA I L Anthonisen, M L Salvador and U Klein Chlamydomonas reinhardtii.atpB gene mRNas stabilize transcripts in the chloroplast of Specific sequence elements in the 5' untranslated regions of rbcL and

ServiceEmail Alerting

click here.right corner of the article or

Receive free email alerts when new articles cite this article - sign up in the box at the top

http://rnajournal.cshlp.org/subscriptions go to: RNATo subscribe to


http://rnajournal.cshlp.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=rna;7/7/1024&return_type=article&return_url=http://rnajournal.cshlp.org/content/7/7/1024.full.pdf

http://rnajournal.cshlp.org/cgi/adclick/?ad=41176&adclick=true&url=http%3A%2F%2Fwww.exiqon.com%2Fls%2FDocuments%2FScientific%2FExiqonInVivoGuidelines.pdf%3Futm_source%3DCSHL%26utm_medium%3Dbanner%26utm_content%3DRNA-2016-07%26utm_campaign%3DFA

http://rnajournal.cshlp.org/subscriptions

