BACKGROUND
chloroplasts originated around  <dig>  billion years ago via an endosymbiotic event where a primitive eukaryote engulfed an ancestor of modern-day cyanobacteria.
subsequently, massive gene transfer to the nucleus occurred, resulting in a highly reduced plastid genome of 120- <dig> kb that possesses ~ <dig> genes.
these remaining genes are mostly organized into clusters, and their expression requires a combination of prokaryotic and eukaryotic-like post-transcriptional events including maturation of polycistronic transcripts, splicing, rna editing, and 5' and 3' end trimming .
these processes are catalyzed by nucleus-encoded proteins, many of which were originally encoded by the chloroplast ancestor  <cit> .
post-transcriptional regulation in both prokaryotes and eukaryotes is also exerted by non-coding rnas , which include antisense rnas  and transcribed intergenic sequences.
the asrnas can act in cis, on the cognizant sense strand transcript, or in trans, targeting one or more distantly-located genes through specific base pairing.
antisense rnas regulate many steps in gene expression including translation initiation, mrna stability, alternative splicing, rna editing, and transcription termination  <cit> .
clear evidence exists that asrnas are prevalent in cyanobacteria, the prokaryote most closely related to chloroplasts.
in prochlorococcus med <dig> and synechocystis sp.
pcc  <dig>  asrnas were found using microarrays, and some were verified by rna blot and shown to be differentially regulated under altered stress and nutrient conditions  <cit> .
functions have been ascribed to two cyanobacterial asrnas, α-fura and isrr, which act in cis to occlude a ribosomal binding site and regulate sense strand transcript accumulation, respectively  <cit> .
thus, the chloroplast progenitor likely used asrnas for gene regulation, suggesting that at least part of this capacity might have been retained in the present-day organelle.
the occurrence of organellar ncrnas has been established experimentally from sequencing of cdna libraries  <cit> .
in one study, ncrnas were abundantly detected in plant mitochondria deficient for polynucleotide phosphorylase , a 3' to 5' exoribonuclease involved in rna maturation and decay, some of which were also found in wild-type  plants  <cit> .
in chloroplasts, sequencing of small rnas identified a number of ncrnas  <cit> .
although their functions were not directly tested, at least some of them appear to be footprints of sequence-specific rna-binding proteins involved in mrna 3' end formation  <cit> .
more recently, an rna antisense to the chloroplast ndhb gene in arabidopsis, nicotiana tabacum and poplar was identified, which was hypothesized to regulate rna maturation or stability  <cit> .
direct evidence for chloroplast asrna function comes primarily from two studies, one in which an ectopically expressed asrna resulted in reduced c-to-u editing in tobacco  <cit> , and a second where a chloroplast genome rearrangement in chlamydomonas led to expression of an asrna that stabilized the corresponding sense strand transcript  <cit> .
the present study focuses on as <dig>  a chloroplast-encoded asrna that is represented by several ests , and was found to be highly abundant in the arabidopsis rnr1- <dig> mutant .
this mutant lacks ribonuclease r , a 3' to 5' exoribonuclease, which is dually targeted to mitochondria and chloroplasts  <cit> , and whose prokaryotic orthologue is involved in rrna maturation and the degradation of structured rnas  <cit> .
the rnr1- <dig> mutant accumulates precursors for several transcripts encoded in the chloroplast ribosomal operon, but is particularly deficient in the accumulation of both precursor and mature forms of 5s rrna, which is encoded by the rrn <dig> gene that lies just upstream of trnr, which encodes trnaarg.
because as <dig> is antisense to the 5s-trnr region, and because rnr1- <dig> overaccumulates as <dig> and underaccumulates the complementary 5s rrna, we postulated that as <dig> might regulate 5s rrna processing or stability.
here we have overexpressed as <dig> in tobacco  chloroplasts to determine its in vivo function in the absence of any pleiotropic effects caused by rnr <dig> deficiency.
the results show that overepxressing as <dig> leads to a slow-growth phenotype and decreased 5s rrna stability, suggesting a possible function for as <dig> in wt chloroplasts.
RESULTS
creation of tobacco chloroplast transformants
transplastomic tobacco plants were obtained using the vector pas5ox, which resulted in the overexpression of as <dig> .
this vector targets the trni-trna region in the chloroplast ribosomal  operon located in the inverted repeat region through homologous recombination, and relies on readthrough from the upstream prrn promoter for as <dig> transcription.
this insertion site is well-characterized and has not been reported to affect plant growth and development .
the selectable marker aada possesses its own promoter and is transcribed in tandem with the rrn operon.
as shown below, this leads to enhanced transcription of genes downstream of aada.
the as <dig> sequence expressed from the transformation vector is the antisense strand extending from just downstream of the trnr gene, to the 3' end of rrn <dig>  endogenous as <dig> is also expressed from this operon, albeit on the opposite strand.
chloroplast transformants were identified by growth on selective medium and pcr .
after 3- <dig> rounds of regeneration, the transgenic lines were further validated by dna gel blot after restriction digest with xhoi and hindiii .
the xhoi site is within the trni intron, while hindiii cleaves in the middle of the as <dig> transgene and just downstream of trna.
two independent as <dig> overexpression  lines were confirmed , with expected bands of  <dig>  and  <dig>  kb .
surprisingly, both lines retained a weak band at  <dig>  kb, corresponding to a wt-like size.
this is likely due to a nuclear-encoded fragment, rather than a plastome sequence, and has been seen in other chloroplast transformants targeting the rrn operon and other regions  <cit> .
furthermore, an identical pattern was observed after the plants had been self-crossed, which normally resolves any residual heteroplasmy.
to verify that increased as <dig> expression was occurring in the as5ox lines, we used quantitative rt-pcr .
after normalization to two nuclear control mrnas, we found that the as5ox lines accumulated >500-fold more as <dig>  confirming the expected transgene expression.
plant growth is slowed in the as5ox lines
growth was compared between as5ox- <dig>  as5ox- <dig> and wt tobacco grown in the greenhouse.
the as5ox lines exhibited slower growth beginning  <dig> days after germination, which resulted in delayed flowering time .
forty days after germination, we observed that internode  <dig> was not significantly reduced in length, while internode  <dig> was shorter.
in addition, the stem circumference at both internodes was significantly reduced .
the differences in internode length and width resulted in shorter plants with lighter shoots after  <dig> days.
shoot fresh weight at maturity was reduced by about 10% compared to the wt, which can likely be ascribed to reduced stem diameter, as the plant height at maturity did not differ .
leaf  <dig> and leaf  <dig> dimensions and weight on a per area basis were measured  <dig> days after germination.
leaf  <dig>  near the base of the plant, was not significantly altered in length, width or fresh weight, while the dry mass was slightly increased in the as5ox plants.
however, leaf  <dig> was significantly smaller in both length and width, while unit area weight was unaffected.
lastly, total chlorophyll was reduced by ~10% in the as5ox lines at maturity, corresponding to a slightly pale-green leaf phenotype.
taken together, these data indicate that the overall vigor of the as5ox lines is diminished, especially with respect to stem elongation.
results represent an average ± the standard error of measurements taken  <dig> days after germination  or at maturity .
internode and leaf numbers were counted starting from the base of the plant.
*traits with a significant difference  between both as5ox lines and wt.
as <dig> is present as multiple transcripts
est data from tobacco and its solanaceous relative tomato suggested that as <dig> is present in multiple overlapping forms .
these ests, however, represent partial sequences from size-selected cdnas, and are derived from polyadenylated transcripts, which are inherently unstable in chloroplasts  <cit> .
to assess which discrete species might accumulate in wt or transformed plants, a series of overlapping strand-specific probes spanning the 5s-trnr region were used on polyacrylamide gel blots of total leaf rna .
the longest probe, p <dig>  contained the 5s-trnr intergenic region and the trnr coding region.
the resulting banding pattern revealed four as <dig> transcripts of 70- <dig> nt in the wt , and an additional  <dig> nt species in the as5ox lines.
the multiple species may result from endo- and/or exonucleolytic maturation of an as <dig> primary transcript, and/or from specific cleavages of as <dig> bound to a sense rna target.
each of the four as <dig> bands common to all samples overaccumulated in the as5ox lines, suggesting that at least a portion of the transgene transcript was functioning in a manner similar to the endogenous one.
the as5ox lines also accumulated larger transcripts not resolved in the polyacrylamide gel .
additional probes were used to further characterize the as <dig> species.
probe p <dig> contained only the 5s-trnr intergenic region and identified all four wt bands, indicating that the wt1- <dig> bands must contain part of the intergenic region.
however, the wt <dig> band was most strongly detected.
this increased hybridization to the wt <dig> band, as compared to results with p <dig>  may reflect secondary structure differences between probes p <dig> and p <dig>  the as5ox-specific band was not detected with p <dig>  demonstrating that this band contains sequences antisense to the trnr coding region, and possibly additional sequence derived from the transgene.
probe p <dig> spans the proximal part of the 5s-trnr intergenic region.
this probe hybridized more weakly than p <dig> and p <dig>  which may be due to less sequence overlap with the targets, and/or its low  g+c content compared to the full intergenic region .
the strongest hybridization was to wt <dig> and wt <dig>  although wt <dig> was faintly visible.
this suggests that wt <dig> and wt <dig> are fully derived from the region covered by p <dig>  wt <dig> appears to have some overlap with p <dig>  but is likely to be mostly derived from more distal portions of the intergenic sequence, whereas wt <dig> appears to be derived entirely from the distal part of the intergenic region.
it is possible that wt <dig> serves as a precursor for each of the smaller transcripts, but this cannot be directly inferred.
in summary, the as5ox lines overproduce the native as <dig> species and at least one novel small as <dig> molecule.
as5ox lines accumulate processing intermediates of rrn transcripts
because the transgene was inserted between trni and trna, rrn operon transcripts were analyzed by rna gel blot.
the operon encodes four rrnas and three trnas that are transcribed as an ~ <dig>  kb polycistronic precursor from a bacterial-like promoter upstream of rrn <dig> .
a series of endo- and exonucleolytic steps, along with splicing of the trni and trna introns, yield the mature rnas.
the probes shown along the bottom of figure 4a were used to detect any perturbations due to as <dig> transgene insertion or regulation.
when blots were probed for rrn <dig>  mature 16s rrna and a precursor were detected .
this precursor is created by endonucleolytic cleavage in the rrn16-trni intergenic region, leaving a 3' extension that is processed exonucleolytically  <cit> .
abundance of the mature and precursor rrn <dig> transcripts was slightly reduced in the as5ox lines.
the trni probe detected mature trnaile , a large polycistronic intermediate , and the ~ <dig>  kb unspliced trni monocistron.
in the as5ox lines, two additional polycistronic precursors accumulated, which appear to contain transgene sequences because they are not present in the wt and comigrate with as5ox-specific as <dig> bands.
unspliced trni was invariant, and accumulation of mature trnaile decreased by ~50% in the as5ox lines.
decreased efficiency of endonucleolytic cleavage at the 3' end of trni, near the transgene insertion site, may be contributing to the reduced formation of mature trnaile and increased accumulation of polycistronic intermediates.
the as <dig> probe identified several transcripts.
the largest polycistronic transcript appears to comigrate with the wt trni polycistron, in addition to the two as5ox-specific trni bands mentioned above.
other less-abundant bands were apparent, the smallest of which migrated near trnaile and represents the abundant as5ox-specific band detected with probe p <dig> in figure 3b.
we next probed for the aada selectable marker transcript.
the aada cassette generates a monocistronic mrna of  <dig>  kb, which was by far the most abundant species.
however, increased exposure revealed both longer bands and one shorter band.
the former appear to be cotranscripts with trni and the as <dig> transgene, whereas the shorter band appears to be a cotranscript with only as <dig>  thus, as5ox plants accumulate a variety of as5-containing transcripts, some of which are the same size as endogenous species and others that are transgene-specific products.
the implications of these findings for possible as <dig> biological activity are considered in the discussion.
the lower part of figure 4b shows probes for the rrn operon downstream of aada.
like trni, the trna probe revealed accumulation of additional precursors in the as5ox lines, however no effect on trnaala accumulation was observed.
the two precursor transcripts also found in wt were a doublet at  <dig>  kb and a  <dig>  kb species, the latter corresponding to unspliced trna.
each of these rnas overaccumulated compared to the wt.
at least five additional bands were detected in the as5ox lines, which appear to be cotranscripts with aada.
analysis of rrn <dig> transcripts revealed seven major species.
this complex pattern is due to accumulation of rrn23-rrn <dig>  processing intermediates and hidden breaks within the rrn <dig> transcript  <cit> .
there were no size differences between the wt and as5ox lines and only minor quantitative differences, suggesting that there was no accumulation of aada cotranscripts.
similarly, accumulation of mature  <dig> s rrna was not affected.
finally, strand-specific probes were used to assess processing and accumulation of 5s rrna and trnaarg.
maturation of these species involves the formation of a transient 5s-trnr intermediate that is cleaved by rnase p to form the 5' end of trnaarg, followed by a second endonuclease cleavage in the 5s-trnr intergenic region near the mature 5s 3' end  <cit> .
the pre-5s rrna is further matured by the 3' to 5' exonucleolytic activity of rnr <dig> and possibly pnpase  <cit> .
when probed for 5s rrna, accumulation was similar between the wt and as5ox lines.
in contrast, trnaarg overaccumulated in the as5ox lines.
as discussed below, this results from increased transcription from the aada promoter.
no precursors were observed for either rrn <dig> or trnr, including when analyzed following high-resolution polyacrylamide gel electrophoresis , indicating that maturation of their 5' and 3' ends was largely unaffected.
because the rrn <dig> and trnr transcripts were the most likely targets of regulation by the overexpressed asrna, qrt-pcr was used to quantify their accumulation relative to two nuclear transcripts .
as suggested by the gel blots , there was only a small increase in rrn <dig> accumulation in the as5ox lines, while the trnr transcript increased three-fold.
overall, the results in figures  <dig>   <dig> show that some rrn operon transcripts accumulated differently in the as5ox lines.
this may be due to differences in transcription rates, rna processing, and/or rna stability.
increased transcription of 5s-trnr in the as5ox plants
we had hypothesized that as <dig> regulates rrn <dig> processing and/or stability, and we observed that in the as5ox lines 5s rrna accumulation was unaffected whereas trnaarg was three-fold more abundant.
one explanation to account for increased trnaarg accumulation was enhanced transcription due to the upstream aada promoter.
this would a priori affect rrn <dig>  rrn <dig> , rrn <dig> and trnr, but not genes upstream of aada, such as rrn <dig>  to determine relative transcription rates in the wt and as5ox lines, a run-on assay was performed.
purified chloroplasts were labeled with α-32p-utp for  <dig> min, after which labeled rnas were used to probe a slot blot containing  <dig> and  <dig> ng of pcr products from rrnas, the photosystem ii d <dig> protein-coding gene , and the chloroamphenicol acetyltransferase gene  as a negative control .
because the signals did not increase in the  <dig> ng slots, we concluded that the target dna was in excess over the probe.
we also noted a weak signal from the cat gene, which was considered background noise and was subtracted from subsequent calculations.
signal intensities were normalized to psba, whose transcription should be unaffected in as5ox lines.
the analysis showed that rrn <dig> transcription varied less than 10%, with the slight decrease mirroring the rna gel blot result .
as rrn <dig> is proximal to the transgene promoter, a different transcription rate was not expected.
when rrn <dig> was examined, the transcription rate was almost doubled in the as5ox lines, and 5s-trnr transcription increased four-fold.
the disparity between rrn <dig> and 5s-trnr results may be attributed to the low signal to noise ratio for the latter in the wt, making the measurement less accurate.
in any event, these results show that while transcription of rrn <dig> and trnr are increased in the as5ox lines, only trnaarg increases in abundance, indicating that 5s rrna is less stable in the as5ox lines.
the unstable species could either be the mature form or a precursor with a 3' extension following trnr excision.
in summary, increased transcription of 5s rrna masks the instability imparted by overexpression of as <dig>
5s rrna polysome association decreases in as5ox lines
the as5ox lines have a slow-growth phenotype and decreased 5s rrna stability.
if as <dig> were destabilizing mature or precursor 5s rrna, it might also interfere with its incorporation into mature ribosomes.
polysome analysis was therefore conducted to assess ribosomal association of 5s rrna and two representative mrnas.
as shown in figure  <dig>  polysome fractions were analyzed for rrn <dig>  rbcl, psba and as <dig>  the rrn <dig> probe revealed that a large portion of mature 5s rrna is not associated with polysomes in either wt or as5ox lines.
this was expected, since 5s rrna exists in a dynamic relationship with the 50s ribosomal subunit, an association thought to influence ribosome performance  <cit> .
in the as5ox lines, however, there was a small but distinctive and reproducible shift of 5s rrna towards less dense fractions , with about one quarter being present in the polysome fractions .
this may be an indirect influence of as <dig> overaccumulation .
polysomal loading of the rbcl and psba mrnas were investigated to identify any effect on chloroplast translational activity.
as was the case for 5s rrna, the rbcl mrna shifted modestly towards the nonpolysomal fractions in the as5ox lines, suggesting that its translation rate had decreased, in keeping with their slower growth phenotype.
the polysomal loading of psba mrna was not appreciably altered between wt and as5ox lines.
however, this transcript is mostly nonpolysomal even in wt plants, except under conditions where psii repair is induced .
finally, the as <dig> transcript itself was examined.
considering the data shown in figure 4b for total rna, we expected to observe approximately six bands between  <dig> nt and  <dig>  kb, with the smaller species being only visible when polyacrylamide gels were used .
figure  <dig> shows that the vast majority of as <dig> rnas in the as5ox lines migrated in the nonpolysomal fractions.
this result was largely expected for an ncrna, however we noted that a portion of the longer as <dig> transcripts were found in heavier fractions.
intriguingly, their accumulation pattern roughly parallels that of 5s rrna.
this raises the possibility that as <dig> may bind to rrn <dig> that is incorporated into polysomes, although the hybridization results do not demonstrate that the two rnas are actually associated.
the  <dig> nt as <dig> band found only in the as5ox lines was in fraction  <dig>  at the top of the gradient.
the other small as <dig> bands are not visible in this gel system.
faint higher molecular weight bands visible on the wt blot were judged to be rrna artifacts based on their migrations.
discussion
ncrnas have been detected in both mitochondria and chloroplasts, yet there is little knowledge of their functional significance.
in this study, the chloroplast asrna as <dig> was investigated for possible cis-acting regulation of rrn <dig> and trnr.
we used the approach of chloroplast transformation to overexpress as <dig>  a strategy not previously used for a natural chloroplast asrna.
however, overexpression of nuclear-encoded mirna is routinely used to confirm mirna targets .
while transgenes can create pleiotropic effects, we were able to correlate increased as <dig> expression with decreased 5s rrna stability and slightly reduced polysome association.
while the underlying mechanism remains to be elucidated, we speculate that as <dig> may restrict 5s rrna ribosomal incorporation and/or pair with its precursor, targeting the duplex for degradation via a double-stranded endoribonuclease.
rna processing factors for the rrn operon may be limited in as5ox lines
when as <dig> was expressed from within the rrn operon, several probes showed varied accumulation of precursor transcripts or processing intermediates compared to the wt.
some of these disparities may have arisen because rna processing capacity had been exceeded, a phenomenon that has previously been observed.
transgenes inserted into the chloroplast rrn operon have been shown to accumulate >100-fold more rna than an analogous nuclear  transformant due to plastid genome polyploidy  <cit> .
excess rna produced by a chloroplast transgene would unlikely stimulate any increase in the nuclear-encoded and chloroplast-targeted proteins necessary for its maturation.
for example, when the clpp 5' utr was overexpressed in tobacco chloroplasts a reduction in endogenous mature clpp mrna was observed due to the limited availability of a clpp-specific mrna maturation factor  <cit> .
in this study, we observed a several-fold increase in transcription rates distal to the aada insertion, which is substantial given that the rrn promoter is already the strongest in the chloroplast  <cit> .
the most strongly affected gene in this region was trna, most likely due to its proximity to the aada promoter.
extra precursor accumulation from the rrn operon, however, probably does not contribute to the slow-growth phenotype of as5ox lines.
previous studies where transgenes were targeted to the rrn operon also resulted in substantial accumulation of polycistronic transcripts, but little effect on plant development and chloroplast protein expression was noted, other than the intended effects of the transgenes, such as increasing tolerance to abiotic stress  <cit> .
however, in one case plant development was delayed  <cit> .
mature trnaile accumulation is decreased somewhat in the as5ox lines, while trnaala was not affected.
we considered whether trnaile deficiency could explain the polysome shift  and slow growth phenotype.
we concluded this is unlikely because the chloroplast encodes a second trnaile isoacceptor  within the inverted repeat region that could compensate for the decrease in the affected trnaile-gau using wobble base pairing, which is common in chloroplasts  <cit> .
the accumulation of trna precursors can probably be ascribed to transgene sequences that interfere with rna structures recognized by processing enzymes.
multiple as5-containing precursor transcripts also accumulated in the transgenic lines, while a family of smaller as <dig> transcripts  were detected by rna gel blot for both wt and as5ox plants.
the precursor transcripts are polycistronic intermediates, while the smaller transcripts may be a result of as <dig> maturation or degradation.
the as5-containing polycistronic transcripts appear to be poor substrates for chloroplast rna processing enzymes, although the smaller species exhibited a 3- to 10-fold greater accumulation in the as5ox lines than in the wt .
this is far less than the qrt-pcr results, which showed a >500-fold overaccumulation of as <dig> in the transgenic lines due to quantification of the very abundant polycistronic precursors in addition to the smaller as <dig> species.
these longer rnas could have less biological activity than the small molecules in terms of 5s rrna binding and/or destabilization, although a small proportion of them was found in the heavier fractions of polysome gradients .
5s rrna stability is decreased in transgenic plants
accumulation of mature rrna transcripts differed in the as5ox lines.
it was expected and observed that transcription of rrn <dig> and rrn <dig> would increase due to the position of the transgene promoter, and that rrn <dig> would be unaffected.
however, there was only a minor  increase in mature 23s rrna, no difference in  <dig> s and 5s rrnas, and a small decrease in 16s transcripts.
the mechanisms surrounding rrna degradation and ribosome copy control in plant organelles are poorly understood, although in bacteria it has been established that rrnas accumulate in a molar ratio with their ribosomal protein counterparts, and if one or the other is in excess they are targeted for degradation .
bacterial rrna turnover also occurs as a way to regulate translation during slow growth and under certain stress conditions, and to prevent the accumulation of misassembled ribosomes.
the variability in mature rrna accumulation in the as5ox lines is likely due to post-transcriptional mechanisms that equalize rrna stoichiometry by degrading excess transcripts according to the bacterial paradigm.
to this end, we speculate that as <dig> may be regulating 5s accumulation, leading to degradation of excess 23s and  <dig> s rrnas.
crosstalk between chloroplast rrnas was previously observed in the tomato dcl mutant, whose primary deficiency is in  <dig> s rrna, but also exhbited decreased polysomal loading of 5s rrna  <cit> .
native as <dig> may be processed in vivo
multiple as <dig> species were detected in both wt and as5ox lines.
among ncrnas, the existence of multiple transcripts is relatively common for eukaryotic mirnas and their precursors  <cit> .
the transcript profiles of organellar ncrnas, however, have been characterized in only a few cases.
in the case of chloroplast ncrnas, both very small  and longer  transcripts have been verified by gel blots.
in the latter case, three transcripts accumulated from the ndhb antisense strand.
as <dig> could be produced by transcriptional read-through from trnn, and/or from a promoter between trnn and as <dig>  either would create longer precursors that could give rise to the observed species.
further evidence that as <dig> is processed comes from the est database, which shows variable 5' and 3' ends from tomato as <dig> transcripts , something that is mirrored when as <dig> ests are extracted for arabidopsis .
however, ests are often derived from protocols that exclude smaller transcripts , so as <dig> transcripts such as the  <dig> nt species detected by rna gel blot  would not be found even if it were polyadenylated.
the  <dig> nt as <dig> species is derived from the 5s-trnr intergenic region near the mature 5s rrna 3' end, a region previously shown to be a site for endonuclease cleavage during 5s rrna maturation  <cit> .
this raises the possibility that some as <dig> bands could be derived from cleavage of a longer as <dig> species bound to its putative 5s rrna target.
both 5s rrna and the 5s-trnr intergenic region are predicted to form multiple stem-loops  <cit> , which would interfere with full-length sense-antisense duplex formation and leave single-stranded regions available for ribonucleolytic cleavage, or promote cleavage by an rnase iii-like activity.
partial duplexing between exposed loops or bulges is common for prokaryotic ncrnas and their targets  <cit> .
one example is binding of dsra to its mrna target, rpos.
this creates an rnase iii processing site, which, following cleavage, produces stable transcripts of each  <cit> .
some as <dig> transcripts may be degradation intermediates unrelated to 5s rrna interactions.
in prokaryotes, both bound and unbound ncrnas are regulated by multiple ribonucleases, including rnase e, pnpase, rnase ii and rnase iii.
for example, the bacterial ncrna rnai degradation intermediate is visible by rna blot after rnase e cleavage near the 5' end.
following this, the transcript is polyadenylated and degraded by rnase ii and pnpase  <cit> .
similarly, multiple transcripts are observed for the salmonella typhimurium ncrnas csra, csrb, mica, and sral, which were determined to be degradation intermediates  <cit> .
as <dig> may regulate 5s rrna
as discussed above, our results lead to the hypothesis that at least a portion of overexpessed as <dig> is binding to pre- and/or mature 5s rrna to inhibit its maturation or incorporation into ribosomes, ultimately targeting it for degradation via an rnase e or rnase iii-dependent pathway.
rnase e is present in chloroplasts  <cit> , and one rnase iii-like protein has been detected to date  <cit> .
additional members of the prokaryotic rnase iii family have also been predicted  <cit> .
based on bacterial data, 5s rrna is thought to stabilize the 50s subunit, enhance peptidyl transferase activity, and assist in trna binding  <cit> .
in e. coli, sequential deletion of 5s rrna genes resulted in a slower growth rate  <cit> , which parallels the slower growth of the as5ox lines where 5s rrna availability may be limited by as <dig>  mature 5s rrna binds to ribosomal proteins l <dig> and l <dig>  and this complex is incorporated into the pre-50s subunit, a process that may be facilitated by the rna-binding proteins csp41a and csp41b  <cit> .
excess as <dig> may be a competitive inhibitor of l <dig> and l <dig>  preventing ribosomal incorporation.
our working hypothesis is that as <dig> is part of a quality/quantity control mechanism for 5s rrna.
while 5s rrna has not been proposed as a regulatory target in chloroplasts, other examples do exist.
in the case of nuclear-encoded 5s rrna in xenopus oocytes, the ro protein associates specifically with 5s sequences that have extra 3' nucleotides and/or point mutations.
these faulty 5s sequences are eventually degraded, and it is thought that binding of ro acts as a chaperone for target recognition by ribonucleases  <cit> .
a second regulatory mechanism occurs in arabidopsis via a heterochromatic sirna, sir <dig>  in this case, aberrant 5s transcripts extending into an intergenic spacer region are cleaved into sirnas.
these sirnas regulate the methylation status of the 5s rdna to prevent further accumulation of long 5s transcripts  <cit> .
while these particular modes of 5s regulation could not occur in chloroplasts, they do suggest that organisms have evolved ways of detecting excess and/or faulty 5s rrna.
CONCLUSIONS
as <dig> is one of the first natural chloroplast-encoded asrnas to be investigated for a possible cis regulatory role in gene expression.
this function is commonly observed for both prokaryotic and eukaryotic ncrnas.
overexpression of as <dig> led to slower growth, which we interpret as a pleiotropic effect of its interaction with 5s rrna.
as <dig> may interact dynamically with 5s rrna as influenced by the accumulation of other ribosomal transcripts and proteins.
we hypothesize that the role of as <dig> is to prevent the accumulation of misprocessed 5s rrna, as well as to control its stoichiometry.
further studies are needed to determine the enzymes involved in the maturation and turnover of as <dig>  and other chloroplast-encoded asrnas.

 chloroplasts originated around billion years ago via an endosymbiotic event where a primitive eukaryote engulfed an ancestor of modern-day cyanobacteria. subsequently, massive gene transfer to the nucleus occurred, resulting in a highly reduced plastid genome of 120- kb that possesses ~ genes. these remaining genes are mostly organized into clusters, and their expression requires a combination of prokaryotic and eukaryotic-like post-transcriptional events including maturation of polycistronic transcripts, splicing, rna editing, and 5' and 3' end trimming. these processes are catalyzed by nucleus-encoded proteins, many of which were originally encoded by the chloroplast ancestor. post-transcriptional regulation in both prokaryotes and eukaryotes is also exerted by non-coding rnas, which include antisense rnas and transcribed intergenic sequences. the asrnas can act in cis, on the cognizant sense strand transcript, or in trans, targeting one or more distantly-located genes through specific base pairing. antisense rnas regulate many steps in gene expression including translation initiation, mrna stability, alternative splicing, rna editing, and transcription termination. clear evidence exists that asrnas are prevalent in cyanobacteria, the prokaryote most closely related to chloroplasts. in prochlorococcus med and synechocystis sp. pcc asrnas were found using microarrays, and some were verified by rna blot and shown to be differentially regulated under altered stress and nutrient conditions. functions have been ascribed to two cyanobacterial asrnas, α-fura and isrr, which act in cis to occlude a ribosomal binding site and regulate sense strand transcript accumulation, respectively. thus, the chloroplast progenitor likely used asrnas for gene regulation, suggesting that at least part of this capacity might have been retained in the present-day organelle. the occurrence of organellar ncrnas has been established experimentally from sequencing of cdna libraries. in one study, ncrnas were abundantly detected in plant mitochondria deficient for polynucleotide phosphorylase, a 3' to 5' exoribonuclease involved in rna maturation and decay, some of which were also found in wild-type plants. in chloroplasts, sequencing of small rnas identified a number of ncrnas. although their functions were not directly tested, at least some of them appear to be footprints of sequence-specific rna-binding proteins involved in mrna 3' end formation. more recently, an rna antisense to the chloroplast ndhb gene in arabidopsis, nicotiana tabacum and poplar was identified, which was hypothesized to regulate rna maturation or stability. direct evidence for chloroplast asrna function comes primarily from two studies, one in which an ectopically expressed asrna resulted in reduced c-to-u editing in tobacco, and a second where a chloroplast genome rearrangement in chlamydomonas led to expression of an asrna that stabilized the corresponding sense strand transcript. the present study focuses on as a chloroplast-encoded asrna that is represented by several ests, and was found to be highly abundant in the arabidopsis rnr1- mutant. this mutant lacks ribonuclease r, a 3' to 5' exoribonuclease, which is dually targeted to mitochondria and chloroplasts, and whose prokaryotic orthologue is involved in rrna maturation and the degradation of structured rnas. the rnr1- mutant accumulates precursors for several transcripts encoded in the chloroplast ribosomal operon, but is particularly deficient in the accumulation of both precursor and mature forms of 5s rrna, which is encoded by the rrn gene that lies just upstream of trnr, which encodes trnaarg. because as is antisense to the 5s-trnr region, and because rnr1- overaccumulates as and underaccumulates the complementary 5s rrna, we postulated that as might regulate 5s rrna processing or stability. here we have overexpressed as in tobacco chloroplasts to determine its in vivo function in the absence of any pleiotropic effects caused by rnr deficiency. the results show that overepxressing as leads to a slow-growth phenotype and decreased 5s rrna stability, suggesting a possible function for as in wt chloroplasts.  creation of tobacco chloroplast transformants transplastomic tobacco plants were obtained using the vector pas5ox, which resulted in the overexpression of as. this vector targets the trni-trna region in the chloroplast ribosomal operon located in the inverted repeat region through homologous recombination, and relies on readthrough from the upstream prrn promoter for as transcription. this insertion site is well-characterized and has not been reported to affect plant growth and development. the selectable marker aada possesses its own promoter and is transcribed in tandem with the rrn operon. as shown below, this leads to enhanced transcription of genes downstream of aada. the as sequence expressed from the transformation vector is the antisense strand extending from just downstream of the trnr gene, to the 3' end of rrn endogenous as is also expressed from this operon, albeit on the opposite strand. chloroplast transformants were identified by growth on selective medium and pcr. after 3- rounds of regeneration, the transgenic lines were further validated by dna gel blot after restriction digest with xhoi and hindiii. the xhoi site is within the trni intron, while hindiii cleaves in the middle of the as transgene and just downstream of trna. two independent as overexpression lines were confirmed, with expected bands of and kb. surprisingly, both lines retained a weak band at kb, corresponding to a wt-like size. this is likely due to a nuclear-encoded fragment, rather than a plastome sequence, and has been seen in other chloroplast transformants targeting the rrn operon and other regions. furthermore, an identical pattern was observed after the plants had been self-crossed, which normally resolves any residual heteroplasmy. to verify that increased as expression was occurring in the as5ox lines, we used quantitative rt-pcr. after normalization to two nuclear control mrnas, we found that the as5ox lines accumulated >500-fold more as confirming the expected transgene expression. plant growth is slowed in the as5ox lines growth was compared between as5ox- as5ox- and wt tobacco grown in the greenhouse. the as5ox lines exhibited slower growth beginning days after germination, which resulted in delayed flowering time. forty days after germination, we observed that internode was not significantly reduced in length, while internode was shorter. in addition, the stem circumference at both internodes was significantly reduced. the differences in internode length and width resulted in shorter plants with lighter shoots after days. shoot fresh weight at maturity was reduced by about 10% compared to the wt, which can likely be ascribed to reduced stem diameter, as the plant height at maturity did not differ. leaf and leaf dimensions and weight on a per area basis were measured days after germination. leaf near the base of the plant, was not significantly altered in length, width or fresh weight, while the dry mass was slightly increased in the as5ox plants. however, leaf was significantly smaller in both length and width, while unit area weight was unaffected. lastly, total chlorophyll was reduced by ~10% in the as5ox lines at maturity, corresponding to a slightly pale-green leaf phenotype. taken together, these data indicate that the overall vigor of the as5ox lines is diminished, especially with respect to stem elongation. results represent an average ± the standard error of measurements taken days after germination or at maturity. internode and leaf numbers were counted starting from the base of the plant. *traits with a significant difference between both as5ox lines and wt. as is present as multiple transcripts est data from tobacco and its solanaceous relative tomato suggested that as is present in multiple overlapping forms. these ests, however, represent partial sequences from size-selected cdnas, and are derived from polyadenylated transcripts, which are inherently unstable in chloroplasts. to assess which discrete species might accumulate in wt or transformed plants, a series of overlapping strand-specific probes spanning the 5s-trnr region were used on polyacrylamide gel blots of total leaf rna. the longest probe, p contained the 5s-trnr intergenic region and the trnr coding region. the resulting banding pattern revealed four as transcripts of 70- nt in the wt, and an additional nt species in the as5ox lines. the multiple species may result from endo- and/or exonucleolytic maturation of an as primary transcript, and/or from specific cleavages of as bound to a sense rna target. each of the four as bands common to all samples overaccumulated in the as5ox lines, suggesting that at least a portion of the transgene transcript was functioning in a manner similar to the endogenous one. the as5ox lines also accumulated larger transcripts not resolved in the polyacrylamide gel. additional probes were used to further characterize the as species. probe p contained only the 5s-trnr intergenic region and identified all four wt bands, indicating that the wt1- bands must contain part of the intergenic region. however, the wt band was most strongly detected. this increased hybridization to the wt band, as compared to results with p may reflect secondary structure differences between probes p and p the as5ox-specific band was not detected with p demonstrating that this band contains sequences antisense to the trnr coding region, and possibly additional sequence derived from the transgene. probe p spans the proximal part of the 5s-trnr intergenic region. this probe hybridized more weakly than p and p which may be due to less sequence overlap with the targets, and/or its low g+c content compared to the full intergenic region. the strongest hybridization was to wt and wt although wt was faintly visible. this suggests that wt and wt are fully derived from the region covered by p wt appears to have some overlap with p but is likely to be mostly derived from more distal portions of the intergenic sequence, whereas wt appears to be derived entirely from the distal part of the intergenic region. it is possible that wt serves as a precursor for each of the smaller transcripts, but this cannot be directly inferred. in summary, the as5ox lines overproduce the native as species and at least one novel small as molecule. as5ox lines accumulate processing intermediates of rrn transcripts because the transgene was inserted between trni and trna, rrn operon transcripts were analyzed by rna gel blot. the operon encodes four rrnas and three trnas that are transcribed as an ~ kb polycistronic precursor from a bacterial-like promoter upstream of rrn. a series of endo- and exonucleolytic steps, along with splicing of the trni and trna introns, yield the mature rnas. the probes shown along the bottom of figure 4a were used to detect any perturbations due to as transgene insertion or regulation. when blots were probed for rrn mature 16s rrna and a precursor were detected. this precursor is created by endonucleolytic cleavage in the rrn16-trni intergenic region, leaving a 3' extension that is processed exonucleolytically. abundance of the mature and precursor rrn transcripts was slightly reduced in the as5ox lines. the trni probe detected mature trnaile, a large polycistronic intermediate, and the ~ kb unspliced trni monocistron. in the as5ox lines, two additional polycistronic precursors accumulated, which appear to contain transgene sequences because they are not present in the wt and comigrate with as5ox-specific as bands. unspliced trni was invariant, and accumulation of mature trnaile decreased by ~50% in the as5ox lines. decreased efficiency of endonucleolytic cleavage at the 3' end of trni, near the transgene insertion site, may be contributing to the reduced formation of mature trnaile and increased accumulation of polycistronic intermediates. the as probe identified several transcripts. the largest polycistronic transcript appears to comigrate with the wt trni polycistron, in addition to the two as5ox-specific trni bands mentioned above. other less-abundant bands were apparent, the smallest of which migrated near trnaile and represents the abundant as5ox-specific band detected with probe p in figure 3b. we next probed for the aada selectable marker transcript. the aada cassette generates a monocistronic mrna of kb, which was by far the most abundant species. however, increased exposure revealed both longer bands and one shorter band. the former appear to be cotranscripts with trni and the as transgene, whereas the shorter band appears to be a cotranscript with only as thus, as5ox plants accumulate a variety of as5-containing transcripts, some of which are the same size as endogenous species and others that are transgene-specific products. the implications of these findings for possible as biological activity are considered in the discussion. the lower part of figure 4b shows probes for the rrn operon downstream of aada. like trni, the trna probe revealed accumulation of additional precursors in the as5ox lines, however no effect on trnaala accumulation was observed. the two precursor transcripts also found in wt were a doublet at kb and a kb species, the latter corresponding to unspliced trna. each of these rnas overaccumulated compared to the wt. at least five additional bands were detected in the as5ox lines, which appear to be cotranscripts with aada. analysis of rrn transcripts revealed seven major species. this complex pattern is due to accumulation of rrn23-rrn processing intermediates and hidden breaks within the rrn transcript. there were no size differences between the wt and as5ox lines and only minor quantitative differences, suggesting that there was no accumulation of aada cotranscripts. similarly, accumulation of mature s rrna was not affected. finally, strand-specific probes were used to assess processing and accumulation of 5s rrna and trnaarg. maturation of these species involves the formation of a transient 5s-trnr intermediate that is cleaved by rnase p to form the 5' end of trnaarg, followed by a second endonuclease cleavage in the 5s-trnr intergenic region near the mature 5s 3' end. the pre-5s rrna is further matured by the 3' to 5' exonucleolytic activity of rnr and possibly pnpase. when probed for 5s rrna, accumulation was similar between the wt and as5ox lines. in contrast, trnaarg overaccumulated in the as5ox lines. as discussed below, this results from increased transcription from the aada promoter. no precursors were observed for either rrn or trnr, including when analyzed following high-resolution polyacrylamide gel electrophoresis, indicating that maturation of their 5' and 3' ends was largely unaffected. because the rrn and trnr transcripts were the most likely targets of regulation by the overexpressed asrna, qrt-pcr was used to quantify their accumulation relative to two nuclear transcripts. as suggested by the gel blots, there was only a small increase in rrn accumulation in the as5ox lines, while the trnr transcript increased three-fold. overall, the results in figures show that some rrn operon transcripts accumulated differently in the as5ox lines. this may be due to differences in transcription rates, rna processing, and/or rna stability. increased transcription of 5s-trnr in the as5ox plants we had hypothesized that as regulates rrn processing and/or stability, and we observed that in the as5ox lines 5s rrna accumulation was unaffected whereas trnaarg was three-fold more abundant. one explanation to account for increased trnaarg accumulation was enhanced transcription due to the upstream aada promoter. this would a priori affect rrn rrn, rrn and trnr, but not genes upstream of aada, such as rrn to determine relative transcription rates in the wt and as5ox lines, a run-on assay was performed. purified chloroplasts were labeled with α-32p-utp for min, after which labeled rnas were used to probe a slot blot containing and ng of pcr products from rrnas, the photosystem ii d protein-coding gene, and the chloroamphenicol acetyltransferase gene as a negative control. because the signals did not increase in the ng slots, we concluded that the target dna was in excess over the probe. we also noted a weak signal from the cat gene, which was considered background noise and was subtracted from subsequent calculations. signal intensities were normalized to psba, whose transcription should be unaffected in as5ox lines. the analysis showed that rrn transcription varied less than 10%, with the slight decrease mirroring the rna gel blot result. as rrn is proximal to the transgene promoter, a different transcription rate was not expected. when rrn was examined, the transcription rate was almost doubled in the as5ox lines, and 5s-trnr transcription increased four-fold. the disparity between rrn and 5s-trnr results may be attributed to the low signal to noise ratio for the latter in the wt, making the measurement less accurate. in any event, these results show that while transcription of rrn and trnr are increased in the as5ox lines, only trnaarg increases in abundance, indicating that 5s rrna is less stable in the as5ox lines. the unstable species could either be the mature form or a precursor with a 3' extension following trnr excision. in summary, increased transcription of 5s rrna masks the instability imparted by overexpression of as 5s rrna polysome association decreases in as5ox lines the as5ox lines have a slow-growth phenotype and decreased 5s rrna stability. if as were destabilizing mature or precursor 5s rrna, it might also interfere with its incorporation into mature ribosomes. polysome analysis was therefore conducted to assess ribosomal association of 5s rrna and two representative mrnas. as shown in figure polysome fractions were analyzed for rrn rbcl, psba and as the rrn probe revealed that a large portion of mature 5s rrna is not associated with polysomes in either wt or as5ox lines. this was expected, since 5s rrna exists in a dynamic relationship with the 50s ribosomal subunit, an association thought to influence ribosome performance. in the as5ox lines, however, there was a small but distinctive and reproducible shift of 5s rrna towards less dense fractions, with about one quarter being present in the polysome fractions. this may be an indirect influence of as overaccumulation. polysomal loading of the rbcl and psba mrnas were investigated to identify any effect on chloroplast translational activity. as was the case for 5s rrna, the rbcl mrna shifted modestly towards the nonpolysomal fractions in the as5ox lines, suggesting that its translation rate had decreased, in keeping with their slower growth phenotype. the polysomal loading of psba mrna was not appreciably altered between wt and as5ox lines. however, this transcript is mostly nonpolysomal even in wt plants, except under conditions where psii repair is induced. finally, the as transcript itself was examined. considering the data shown in figure 4b for total rna, we expected to observe approximately six bands between nt and kb, with the smaller species being only visible when polyacrylamide gels were used. figure shows that the vast majority of as rnas in the as5ox lines migrated in the nonpolysomal fractions. this result was largely expected for an ncrna, however we noted that a portion of the longer as transcripts were found in heavier fractions. intriguingly, their accumulation pattern roughly parallels that of 5s rrna. this raises the possibility that as may bind to rrn that is incorporated into polysomes, although the hybridization results do not demonstrate that the two rnas are actually associated. the nt as band found only in the as5ox lines was in fraction at the top of the gradient. the other small as bands are not visible in this gel system. faint higher molecular weight bands visible on the wt blot were judged to be rrna artifacts based on their migrations. discussion ncrnas have been detected in both mitochondria and chloroplasts, yet there is little knowledge of their functional significance. in this study, the chloroplast asrna as was investigated for possible cis-acting regulation of rrn and trnr. we used the approach of chloroplast transformation to overexpress as a strategy not previously used for a natural chloroplast asrna. however, overexpression of nuclear-encoded mirna is routinely used to confirm mirna targets. while transgenes can create pleiotropic effects, we were able to correlate increased as expression with decreased 5s rrna stability and slightly reduced polysome association. while the underlying mechanism remains to be elucidated, we speculate that as may restrict 5s rrna ribosomal incorporation and/or pair with its precursor, targeting the duplex for degradation via a double-stranded endoribonuclease. rna processing factors for the rrn operon may be limited in as5ox lines when as was expressed from within the rrn operon, several probes showed varied accumulation of precursor transcripts or processing intermediates compared to the wt. some of these disparities may have arisen because rna processing capacity had been exceeded, a phenomenon that has previously been observed. transgenes inserted into the chloroplast rrn operon have been shown to accumulate >100-fold more rna than an analogous nuclear transformant due to plastid genome polyploidy. excess rna produced by a chloroplast transgene would unlikely stimulate any increase in the nuclear-encoded and chloroplast-targeted proteins necessary for its maturation. for example, when the clpp 5' utr was overexpressed in tobacco chloroplasts a reduction in endogenous mature clpp mrna was observed due to the limited availability of a clpp-specific mrna maturation factor. in this study, we observed a several-fold increase in transcription rates distal to the aada insertion, which is substantial given that the rrn promoter is already the strongest in the chloroplast. the most strongly affected gene in this region was trna, most likely due to its proximity to the aada promoter. extra precursor accumulation from the rrn operon, however, probably does not contribute to the slow-growth phenotype of as5ox lines. previous studies where transgenes were targeted to the rrn operon also resulted in substantial accumulation of polycistronic transcripts, but little effect on plant development and chloroplast protein expression was noted, other than the intended effects of the transgenes, such as increasing tolerance to abiotic stress. however, in one case plant development was delayed. mature trnaile accumulation is decreased somewhat in the as5ox lines, while trnaala was not affected. we considered whether trnaile deficiency could explain the polysome shift and slow growth phenotype. we concluded this is unlikely because the chloroplast encodes a second trnaile isoacceptor within the inverted repeat region that could compensate for the decrease in the affected trnaile-gau using wobble base pairing, which is common in chloroplasts. the accumulation of trna precursors can probably be ascribed to transgene sequences that interfere with rna structures recognized by processing enzymes. multiple as5-containing precursor transcripts also accumulated in the transgenic lines, while a family of smaller as transcripts were detected by rna gel blot for both wt and as5ox plants. the precursor transcripts are polycistronic intermediates, while the smaller transcripts may be a result of as maturation or degradation. the as5-containing polycistronic transcripts appear to be poor substrates for chloroplast rna processing enzymes, although the smaller species exhibited a 3- to 10-fold greater accumulation in the as5ox lines than in the wt. this is far less than the qrt-pcr results, which showed a >500-fold overaccumulation of as in the transgenic lines due to quantification of the very abundant polycistronic precursors in addition to the smaller as species. these longer rnas could have less biological activity than the small molecules in terms of 5s rrna binding and/or destabilization, although a small proportion of them was found in the heavier fractions of polysome gradients. 5s rrna stability is decreased in transgenic plants accumulation of mature rrna transcripts differed in the as5ox lines. it was expected and observed that transcription of rrn and rrn would increase due to the position of the transgene promoter, and that rrn would be unaffected. however, there was only a minor increase in mature 23s rrna, no difference in s and 5s rrnas, and a small decrease in 16s transcripts. the mechanisms surrounding rrna degradation and ribosome copy control in plant organelles are poorly understood, although in bacteria it has been established that rrnas accumulate in a molar ratio with their ribosomal protein counterparts, and if one or the other is in excess they are targeted for degradation. bacterial rrna turnover also occurs as a way to regulate translation during slow growth and under certain stress conditions, and to prevent the accumulation of misassembled ribosomes. the variability in mature rrna accumulation in the as5ox lines is likely due to post-transcriptional mechanisms that equalize rrna stoichiometry by degrading excess transcripts according to the bacterial paradigm. to this end, we speculate that as may be regulating 5s accumulation, leading to degradation of excess 23s and s rrnas. crosstalk between chloroplast rrnas was previously observed in the tomato dcl mutant, whose primary deficiency is in s rrna, but also exhbited decreased polysomal loading of 5s rrna. native as may be processed in vivo multiple as species were detected in both wt and as5ox lines. among ncrnas, the existence of multiple transcripts is relatively common for eukaryotic mirnas and their precursors. the transcript profiles of organellar ncrnas, however, have been characterized in only a few cases. in the case of chloroplast ncrnas, both very small and longer transcripts have been verified by gel blots. in the latter case, three transcripts accumulated from the ndhb antisense strand. as could be produced by transcriptional read-through from trnn, and/or from a promoter between trnn and as either would create longer precursors that could give rise to the observed species. further evidence that as is processed comes from the est database, which shows variable 5' and 3' ends from tomato as transcripts, something that is mirrored when as ests are extracted for arabidopsis. however, ests are often derived from protocols that exclude smaller transcripts, so as transcripts such as the nt species detected by rna gel blot would not be found even if it were polyadenylated. the nt as species is derived from the 5s-trnr intergenic region near the mature 5s rrna 3' end, a region previously shown to be a site for endonuclease cleavage during 5s rrna maturation. this raises the possibility that some as bands could be derived from cleavage of a longer as species bound to its putative 5s rrna target. both 5s rrna and the 5s-trnr intergenic region are predicted to form multiple stem-loops, which would interfere with full-length sense-antisense duplex formation and leave single-stranded regions available for ribonucleolytic cleavage, or promote cleavage by an rnase iii-like activity. partial duplexing between exposed loops or bulges is common for prokaryotic ncrnas and their targets. one example is binding of dsra to its mrna target, rpos. this creates an rnase iii processing site, which, following cleavage, produces stable transcripts of each. some as transcripts may be degradation intermediates unrelated to 5s rrna interactions. in prokaryotes, both bound and unbound ncrnas are regulated by multiple ribonucleases, including rnase e, pnpase, rnase ii and rnase iii. for example, the bacterial ncrna rnai degradation intermediate is visible by rna blot after rnase e cleavage near the 5' end. following this, the transcript is polyadenylated and degraded by rnase ii and pnpase. similarly, multiple transcripts are observed for the salmonella typhimurium ncrnas csra, csrb, mica, and sral, which were determined to be degradation intermediates. as may regulate 5s rrna as discussed above, our results lead to the hypothesis that at least a portion of overexpessed as is binding to pre- and/or mature 5s rrna to inhibit its maturation or incorporation into ribosomes, ultimately targeting it for degradation via an rnase e or rnase iii-dependent pathway. rnase e is present in chloroplasts, and one rnase iii-like protein has been detected to date. additional members of the prokaryotic rnase iii family have also been predicted. based on bacterial data, 5s rrna is thought to stabilize the 50s subunit, enhance peptidyl transferase activity, and assist in trna binding. in e. coli, sequential deletion of 5s rrna genes resulted in a slower growth rate, which parallels the slower growth of the as5ox lines where 5s rrna availability may be limited by as mature 5s rrna binds to ribosomal proteins l and l and this complex is incorporated into the pre-50s subunit, a process that may be facilitated by the rna-binding proteins csp41a and csp41b. excess as may be a competitive inhibitor of l and l preventing ribosomal incorporation. our working hypothesis is that as is part of a quality/quantity control mechanism for 5s rrna. while 5s rrna has not been proposed as a regulatory target in chloroplasts, other examples do exist. in the case of nuclear-encoded 5s rrna in xenopus oocytes, the ro protein associates specifically with 5s sequences that have extra 3' nucleotides and/or point mutations. these faulty 5s sequences are eventually degraded, and it is thought that binding of ro acts as a chaperone for target recognition by ribonucleases. a second regulatory mechanism occurs in arabidopsis via a heterochromatic sirna, sir in this case, aberrant 5s transcripts extending into an intergenic spacer region are cleaved into sirnas. these sirnas regulate the methylation status of the 5s rdna to prevent further accumulation of long 5s transcripts. while these particular modes of 5s regulation could not occur in chloroplasts, they do suggest that organisms have evolved ways of detecting excess and/or faulty 5s rrna.  as is one of the first natural chloroplast-encoded asrnas to be investigated for a possible cis regulatory role in gene expression. this function is commonly observed for both prokaryotic and eukaryotic ncrnas. overexpression of as led to slower growth, which we interpret as a pleiotropic effect of its interaction with 5s rrna. as may interact dynamically with 5s rrna as influenced by the accumulation of other ribosomal transcripts and proteins. we hypothesize that the role of as is to prevent the accumulation of misprocessed 5s rrna, as well as to control its stoichiometry. further studies are needed to determine the enzymes involved in the maturation and turnover of as and other chloroplast-encoded asrnas. 
our results suggest that as can modulate 5s rrna levels, giving it the potential to affect chloroplast translation and plant growth. analysis of as transcripts revealed four distinct species in wild-type and as5ox plants, and additional as5ox-specific products. however, run-on transcription showed that the 5s-trnr region was transcribed four-fold more in the as5ox plants compared to wt, indicating that overexpression of as was associated with decreased stability of 5s rrna. in this study, we used nicotiana tabacum to investigate the effect on sense strand transcripts of overexpressing a natural chloroplast asrna, as which is complementary to the region which encodes the 5s rrna and trnaarg. of the corresponding sense strand transcripts, trnaarg overaccumulated several-fold in transgenic plants whereas 5s rrna was unaffected. in addition, polysome analysis of the transformants showed less 5s rrna and rbcl mrna associated with ribosomes. as5-overexpressing plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. more globally, overexpression of asrnas via chloroplast transformation may be a useful strategy for defining their functions. the roles of non-coding rnas in regulating gene expression have been extensively studied in both prokaryotes and eukaryotes, however few reports exist as to their roles in organellar gene regulation.
 the roles of non-coding rnas in regulating gene expression have been extensively studied in both prokaryotes and eukaryotes, however few reports exist as to their roles in organellar gene regulation. evidence for accumulation of natural antisense rnas in chloroplasts comes from the expressed sequence tag database and cdna libraries, while functional data have been largely obtained from artificial asrnas. in this study, we used nicotiana tabacum to investigate the effect on sense strand transcripts of overexpressing a natural chloroplast asrna, as which is complementary to the region which encodes the 5s rrna and trnaarg.  as5-overexpressing plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. analysis of as transcripts revealed four distinct species in wild-type and as5ox plants, and additional as5ox-specific products. of the corresponding sense strand transcripts, trnaarg overaccumulated several-fold in transgenic plants whereas 5s rrna was unaffected. however, run-on transcription showed that the 5s-trnr region was transcribed four-fold more in the as5ox plants compared to wt, indicating that overexpression of as was associated with decreased stability of 5s rrna. in addition, polysome analysis of the transformants showed less 5s rrna and rbcl mrna associated with ribosomes.  our results suggest that as can modulate 5s rrna levels, giving it the potential to affect chloroplast translation and plant growth. more globally, overexpression of asrnas via chloroplast transformation may be a useful strategy for defining their functions. 