BACKGROUND
the ribosome is responsible for the translation of all mrnas into protein.
specific ribosomal protein mutations underlie certain human diseases .
different ribosomal protein mutations also cause different spectra of developmental defects in metazoans and plants  <cit> .
these observations have raised interest in the contributions of the individual ribosomal proteins to translation of different mrnas.
however, few studies have examined the consequences of ribosome defects on translation, and in the vast majority of case studies no genome-wide data are available.
one exception concerns the ribosomal protein of the small subunit, rps <dig>  a mutation in rps <dig> is responsible for cases of human diamond-blackfan anemia.
the rps <dig> mutation causes both inhibition and stimulation of several dozen mrnas, including several that are implicated in the etiology of the disease  <cit> .
in another example, a mutation in mouse rpl <dig> results in a homeotic transformation affecting the ribcage.
the rpl <dig> mutation interferes with translation of homeobox mrnas  <cit> .
genome-wide studies of translation under different stresses and environmental conditions have provided a wealth of information on global translation control in the model plant arabidopsis thaliana <cit> .
however, even in arabidopsis, which has become one of the premier model organisms for translatome research, information on the role of specific translation factors is limited.
the different isoforms of eukaryotic initiation factors eif4e and eif4g, which are components of the mrna cap-binding complex, contribute differentially to translation  <cit> .
the eif <dig> complex is the largest of the initiation factors.
some evidence points toward a role for eif <dig> in promoting the translation of specific 'client’ mrnas  <cit> .
only one global plant translation profile has been obtained from a mutant with a defect in the translation apparatus  <cit> .
in detail, mutations that delete the c-terminus of eif3h compromise translation reinitiation on mrnas containing upstream open reading frames , and also increase the polysome loading of mrnas with long 5′ leader or coding sequences  <cit> .
ribosomal protein rpl <dig>  is a constituent of the cytosolic large 60s subunit.
a mutant of a. thaliana in which one of three rpl24-encoding genes, rpl24b, is deleted , is characterized by developmental pattern defects in the base of the fruit  <cit> .
normal fruit patterning is regulated by uorfs and requires rpl24-dependent translation reinitiation on certain uorf-containing mrnas  <cit> .
the major rpl24b paralog, rpl24a, has also been implicated in the translation reinitiation on mrnas with multiple coding regions  <cit> .
in single-gene reporter gene expression assays of the uorf-containing mrna, atbzip <dig>  the molecular defects in rpl24b resemble those in the eif3h mutant.
the seedling phenotypes of eif3h and rpl24b, while not identical, overlap with respect to vascular development, gynoecium structures, and other organs  <cit> .
these data suggest that rpl24b and eif3h have related functions, although their exact biochemical roles are unclear.
in arabidopsis, all ribosomal proteins are encoded by at least two paralogs, which are typically at least partially redundant  <cit> .
rpl24a  and rpl24b  are expressed at similarly high levels.
the third and final paralog, rpl24c , is expressed at a fourfold lower level, and the protein may be enriched in the nucleolus  <cit> .
rpl <dig> has also been investigated in mouse and yeast.
a mutation in the single mouse rpl <dig> gene, belly spot and tail , is homozygous lethal while heterozygotes show a pleiotropic developmental phenotype  <cit> .
for comparison, a double mutant in the two rpl <dig> genes of the yeast saccharomyces cerevisiae is not lethal  <cit> .
these results suggest that the yeast ribosome can assemble successfully without rpl <dig>  in keeping with rpl <dig> being one of the last proteins to be assembled into the 60s subunit  <cit> .
another specific protein that has been implicated in plant translational control is poly-binding protein   <cit> .
by bridging between the 5′ cap binding complex and the 3′ poly tail, pabp is thought to enhance ribosome recycling and thus translation  <cit> .
in arabidopsis the class ii pabp genes  are the more highly expressed in seedlings and encode the bulk of pabp in seedlings  <cit> .
there are no reports of mrna-specific translational defects for any pab mutant.
for this study, we sought to identify the translational defects in two new types of mutants that affect the translation apparatus.
we report here one of the first global analyses of ribosome occupancy in any organism for a ribosomal protein mutant, arabidopsis rpl24b.
this mutant was chosen because of its functional association with the initiation factor eif3h, for which a mutant translatome is already available  <cit> .
we also report ribosome occupancy data in the arabidopsis pab <dig> pab <dig> double mutant.
this mutant is viable, yet slightly dwarfed, indicative of compromised pabp activity; a pab <dig> pab <dig> pab <dig> triple mutant has not been recovered  <cit> .
the translational defects in the pab mutant were comparatively subtle and restricted to residual mrnas that are expressed highly in late embryogenesis.
in contrast, the rpl24b mutation altered the ribosome occupancy of hundreds of mrnas.
in keeping with the related developmental phenotypes of the rpl24b and eif3h mutations, their translational defects also overlapped.
ribosomal protein  mrnas were the most highly affected functional category.
the ribosome occupancy of nuclear encoded r-proteins was typically stimulated by the rpl24b mutation.
a large fraction of r-proteins, but not all, appear to belong to a single regulon of translational control.
RESULTS
identification of differentially translated genes in a ribosomal protein mutant
seedlings of previously described mutant alleles were grown for the pab <dig> pab <dig> double mutant  <cit> , the eif3h- <dig> mutant  <cit> , and rpl24b   <cit>  .
polysome microarray data from the rpl24b mutant and the pab <dig> pab <dig> mutant were collected using sucrose gradient fractionation of ribonucleoprotein complexes from arabidopsis seedlings and were processed as described in methods.
in parallel, equivalent data from a published experiment  <cit>  on the eif3h mutant were reanalyzed from the hybridization signals using the same procedure as for rpl24b.
the translation state  is defined as the ratio between the mrna signal in the polysomal: non-polysomal fractions and is displayed as a log <dig> value.
for example, an mrna that is 80% polysomal and 20% non-polysomal receives a tl of  <dig>  and a log <dig> tl of + <dig> .
for this reason the tl value is completely independent of the transcript level .
we carefully evaluated many different data processing procedures in order to optimize detection of differentially translated genes .
dtgs between mutant and wild-type were first mined using a stringent false-discovery-rate  cut-off of 5%.
from the entire set of  <dig>  probe sets, we identified  <dig> and  <dig> dtgs for rpl24b and eif3h, respectively.
because this number for eif3h was small compared to the number of dtgs identified in the earlier publication  <cit> , we sought to expand the gene set.
using the  <dig> additional prefiltering methods laid out in the methods, we assembled a non-redundant set of dtgs,  <dig> for rpl24b and  <dig> for eif3h .
comparison of translation defects between rpl24b and eif3h
because translation of atbzip <dig>  auxin response factor <dig>  and arf <dig>  depend on functional eif3h and rpl24b  <cit> , one might hypothesize that eif3h and rpl24b control the ribosome loading of identical sets of client mrnas.
we tested this hypothesis by comparing clusters of genes that are altered at the translation level in eif3h and rpl24b mutants.
among the dtgs in rpl24b or eif3h, the direction and the degree of mistranslation were significantly correlated .
this indicates that translation of many mrnas are coregulated by, or codependent on, rpl24b and eif3h .
remarkably, among all the mrnas that were overtranslated in eif3h , the vast majority had a trend towards overtranslation in the rpl24b dataset ; the same held true for undertranslated mrnas .
this result indicates that a substantial and significant number of arabidopsis mrnas are codependent on eif3h and rpl24b for appropriate translation.
the mrnas that were translationally stimulated in both rpl24b and eif3h mutants were enriched in ribosomal protein mrnas  while the undertranslated ones had no detectable functional bias.
the effects of rpl24b and eif3h on transcript abundance were also correlated  .
because rpl <dig> and eif3h are translation factors, we presume that most of the changes at the transcript level are a consequence, rather than a cause, of alterations in translation.
we also note that the translational defects in the rpl24b mutant are not strongly correlated with changes in total transcript levels, similar as for eif3h <cit>  .
to work out whether there are mrnas that depend on only  <dig> of the  <dig> genes, rpl24b or eif3h, but not on the other, we performed fuzzy k-means clustering on a total of  <dig> dtgs .
two of six clusters, clusters  <dig> and  <dig>  corresponded to the strongly coregulated mrnas.
two other clusters, clusters  <dig> and  <dig>  showed more limited coregulation, that is, a strong response in one mutant and a moderate trend in the other.
the last two clusters, clusters  <dig> and  <dig>  contained mrnas whose response was much stronger in eif3h than in rpl24b.
there were few rpl24b-specific mrnas.
overall, these results underscore the significant overlap between sets of mrnas mistranslated in eif3h and rpl24b mutants.
these new findings bolster the conclusion that rpl24b and eif3h play related roles in eukaryotic gene expression.
translation states in the pab <dig> pab <dig> double mutant
the poly binding proteins are widely thought to stimulate translation in a cellular context by helping to juxtapose 3′ end and 5′ end of mrnas and by assisting with ribosome recycling.
pab <dig>  pab <dig>  and pab <dig> are the three major pabp genes expressed in seedlings and are functionally redundant.
the pab <dig> pab <dig> double mutant is mildly dwarfed  <cit> .
we examined the ribosome occupancy of mrnas in the pab <dig> pab <dig> double mutant seedlings .
overall, the pab <dig> pab <dig> double mutant had few gene-specific alterations in its ribosome occupancy.
the primary fdr-controlled alteration was an approximately twofold increase in ribosome occupancy for a small group of seed storage protein mrnas .
this result is of interest, as seed storage protein mrnas are under translational control in various other plants  <cit> .
less stringent filtering using an alternative ad hoc method to identify dtgs  recovered oleosins, which are additional seed storage mrnas, and late embryogenesis abundant mrnas among the mrnas with increased ribosome loading in pab <dig> pab <dig>  it is notable that pab <dig>  the major remaining pabp mrna in the pab <dig> pab <dig> mutant, was also translationally stimulated.
this result favors the hypothesis that pabp mrnas are subject to negative translational autoregulation via poly stretches in their 5′ untranslated regions   <cit> .
we compared the translational alterations in the rpl24b mutant with those in pab <dig> pab <dig>  not even a weak correlation was detected .
this important result suggests that different perturbations of the translational machinery affect different sets of mrnas.
because most alterations in ribosome occupancy in pab <dig> pab <dig> were small and statistically insignificant on a single-gene basis, we searched for global patterns.
short mrnas were enriched among the mrnas that were translationally inhibited in the pab <dig> pab <dig> mutant .
this trend could be attributed to the length of the coding sequence as well as the length of the 5′ utr, while the length of the 3′ utr contributed little if at all .
analysis of mrna transcript levels in the pab <dig> pab <dig> mutant revealed  <dig> and  <dig> significantly downregulated and upregulated genes at the mrna transcript level.
the transcript levels of pab <dig> and pab <dig> were reduced, as expected given the t-dna insertions in these genes in the pab <dig> pab <dig> strain.
interestingly, among the  <dig> mrnas with >2-fold increased ribosome occupancy in pab <dig> pab <dig> all except  <dig>  are late-embryogenesis mrnas whose level declines up to 1000-fold during seed germination, the developmental period preceding our analysis.
of them,  <dig> were also upregulated at the transcript level in pab <dig> pab <dig>  for pab <dig> pab <dig>  and in contrast to the situation in rpl24b, the correlation between translational and transcript-level stimulation continued among the less strongly stimulated mrnas .
this result suggests that wild-type pabp might couple transcript turnover and translation state for these late embryogenesis mrnas.
overtranslation of ribosomal protein mrnas in rpl24b and eif3h
we performed genome ontology  analyses of the six clusters of rpl24b or eif3h dependent mrnas identified by fuzzy k-means clustering .
clusters  <dig> and  <dig> were both highly enriched for r-protein mrnas.
of note, cytosolic r-protein mrnas are coordinately affected in response to sucrose starvation  <cit> , drought  <cit> , hypoxia stress  <cit> , and unanticipated darkness  <cit> .
however, in each of these stresses, the r-protein mrnas are undertranslated rather than overtranslated.
to detect smaller effects of rpl24b and eif3h on the ribosomal protein mrnas, we adopted the ad hoc filtering method to identify differentially translated genes   <cit> .
the ad hoc method does not control for fdr .
this method enriched even more significantly for r-proteins .
of all the cytosolic r-protein mrnas for which the arrays yielded data , 41% were found in clusters  <dig> to  <dig>  a  <dig> -fold enrichment.
this result suggests that the ad hoc method, which is less conservative than the fdr method, produces fewer false negative calls.
these results, therefore, confirm in more detail that many but not all cytosolic r-protein mrnas depend on rpl24b and eif3h at the translation level and could define a regulon of translational control  <cit> .
mrnas that were affected by eif3h or rpl24b mutations were not generally perturbed by the pab <dig> pab <dig> mutations or by a mutation in the k subunit of eif <dig>  .
we next visualized the r-protein cohort at a gene-specific level .
among mrnas encoding cytosolic r-proteins the pattern of overtranslation in the rpl24b mutant was generally mirrored and enhanced in the eif3h mutant.
in contrast, the pattern in the pab <dig> pab <dig> mutant bore no resemblance to that in rpl24b or eif3h.
the joint overtranslation in rpl24b and eif3h did not generally extend to the translation initiation factors; among the few that were jointly stimulated, we found a few eif <dig> subunits .
the mutation in rpl24b also stimulated the mrna transcript levels of many cytosolic r-protein mrnas, but the changes at the transcript level were not obviously correlated with those at the translation level.
among the mrnas for organellar r-proteins, those encoded in the nucleus were translationally stimulated in a similar fashion in the two mutants, although the trend was weaker than for the nuclear-encoded cytosolic proteins .
in contrast, mrnas for r-proteins that are encoded in the organelles themselves, that is, the plastid or mitochondrion, all had reduced ribosome loading in the rpl24b and eif3h mutants.
the effect on the organellar mrnas must be indirect because rpl <dig> and eif3h function only in the cytosol.
it should be noted that our array data are biased for polyadenylated mrnas.
in the organelles, polyadenylation is a precursor for degradation  <cit> .
thus, our results suggest that, in the wild-type chloroplasts, polyadenylated mrnas remain substantially ribosome loaded, whereas in the mutants, the ribosome loading of the polyadenylated mrnas is reduced.
fine structure analysis of the translation status of ribosomal protein mrnas
because the r-protein mrnas were reliably expressed and showed robust cotranslation among themselves and between rpl24b and eif3h mutants, we characterized their translational coregulation in greater detail.
figure 5b shows that the ribosome occupancy  of the r-protein mrnas in wild-type plants varied dramatically among the different mrnas.
certain extremely short mrnas should have low ribosome occupancy, simply because few ribosomes fit onto the coding sequence of a small mrna.
this was indeed the case for several of the smallest r-proteins .
however, even among the paralogs of one family, it was common for one paralog to be highly translated, and another paralog to be poorly translated .
however, the reproducibility of the ribosome occupancy between the two replicate experiments was excellent.
certain r-protein mrnas have incomplete coding sequences  <cit>  and have been marked as pseudogenes  <cit> .
these mrnas tended to have lower ribosome occupancy , additional file  <dig>
figure 5a shows how ribosome occupancy of the r-protein mrnas was affected in the rpl24b and eif3h mutants.
the following numbers of mrnas cleared statistical significance at fdr < <dig> :  <dig> mrnas that changed ribosome loading in eif3h and  <dig> in rpl24b .
several generalizations are supported by these data.
first, certain mrnas with very high ribosome occupancy in the wild-type  did not increase their tl in the mutant, presumably simply because tl was already close to maximal .
vice versa, mrnas with medium or low ribosome occupancy in the wild-type had an upward trend in the mutant .
second, for many r-protein families, the degree of translational dependence differed between the different paralogs; clear examples include l <dig>  l <dig>  l <dig>  l18a, l <dig>  l35a, p <dig>  sa, s <dig>  s <dig>  s15a, s <dig>  and s <dig>  third, in keeping with the pattern of cotranslation between rpl24b and eif3h described earlier  the paralog with the strongest stimulation in rpl24b often had the strongest stimulation in eif3h.
for examples, see l <dig>  l <dig>  l <dig>  l <dig>  l <dig>  l18a, l <dig>  l <dig>  l <dig>  l <dig>  p <dig>  s <dig>  s <dig>  s <dig>  and s <dig>  clear exceptions were rare.
rpl <dig> presents one of the few exceptions, almost certainly because the rpl24b mrna is truncated by a deletion in the rpl24b mutant  <cit> .
fourth, in a minority of r-protein families all paralogs were slightly reduced in their ribosome loading in the rpl24b mutant.
most of these r-proteins were small proteins of less than  <dig> to 13 kda  <cit> .
examples are l <dig> , l35a , l36a , l37a , l <dig> , s <dig> , s <dig> , s <dig> , s <dig> , and s <dig> .
in contrast, most of the larger r-proteins had at least one paralog that was translationally stimulated in the rpl24b mutant.
on this topic, we note that the translation of these short mrnas was generally normal in the eif3h mutant.
in the eif3h mutant, long mrnas are preferentially inhibited in their ribosome loading, but short mrnas are not  <cit> .
this is one exception from the general rule of correlated translation behavior between the two mutants.
finally, certain r-protein paralogs code for incomplete open reading frames.
of the eight such cases  <cit>  for which we have data , none were translationally stimulated in either of the mutants, in sharp contrast to the general pattern.
indeed, the two mrnas with the strongest translational repression in eif3h, rps12b and rps25c, both encode incomplete orfs and are annotated as pseudogenes in the arabidopsis information resource, release  <dig>  it is unclear why these mrnas are more sensitive to the mutations.
taken together, considering the detailed annotation features just discussed, it appears that the r-protein regulon probably includes more mrnas than was evident from figure  <dig>  while figure  <dig> suggested that certain r-protein mrnas might be excluded from the regulon, detailed inspection of figure  <dig> makes this hypothesis harder to sustain.
of the cytosolic r-proteins above 13 kda, the majority showed translational stimulation in both rpl24b and eif3h for at least one paralog.
paralogs that were stimulated less in the mutants often had a near maximal translation state in the wild-type.
these mrnas should be counted as legitimate members of the r-protein regulon.
other paralogs that bucked the general pattern and should not be considered part of the r-protein regulon code for incomplete r-proteins and rpl24b itself.
molecular features of undertranslated and overtranslated mrnas
given that the rpl24b mutant shared a common set of translationally stimulated and translationally inhibited mrnas with eif3h, we examined whether these mrnas harbored the same features that render some arabidopsis mrnas eif3h-dependent.
these features are uorfs and a long coding region  <cit> .
as shown in additional file 6: figure s <dig>  uorfs only played a weak role in causing poor translation in rpl24b.
in detail, a statistically significant association between uorfs and rpl24b-dependent translation was seen only within the middle range of the distribution , but not for the more strongly rpl24b-dependent mrnas .
the association of orf length with rpl24b-dependence was also weak .
in contrast, for the eif3h dataset, our reanalysis of the earlier data  <cit>  reproduced the conclusion that uorfs correlate with eif3h dependence .
to get a clearer view of this unexpected result, we examined the occurrence of uorfs among the undertranslated genes found in clusters  <dig>   <dig>  and  <dig> .
the mrnas that were clearly codependent on both rpl24b and eif3h, were not enriched for uorfs , while the mrnas that were strongly eif3h-dependent but weakly rpl24b-dependent tended to be the ones with uorfs.
apparently, the rpl24b mutation caused only a minor deficiency in ribosome loading for the uorf-containing mrnas.
this was surprising, because if one measures reporter protein translation downstream from a uorf in the rpl24b mutant, the deficiency in translation can be quite pronounced  <cit> .
in summary, the effects of the mutations in eif3h and rpl24b on ribosome occupancy are related but not identical.
stochastic variation of mrna translation states in wild-type plants
data on mrna translation states can be variable between biological replicates.
we examined the structure of the variation in translation states that we observed among  <dig> different rna samples from wild-type seedlings, collected from seedlings grown on defined agar medium under largely identical experimental conditions.
we focused on the 20% of mrnas with the largest variation in translation state among the replicates, that is, a standard deviation above  <dig>  .
likewise, we identified a group of mrnas with highly variable mrna transcript levels .
gene ontology  of the translationally variable mrnas revealed that the most enriched biological process  was 'response to abiotic stimulus’.
within this category, many different stimuli and stresses were represented and none stood out .
the most enriched cellular compartment was the 'chloroplast’ , and multiple compartments such as 'stroma’ and 'envelope’ , but not 'thylakoid’ , were overrepresented.
the overrepresentation of plasma membrane, apoplast, cell wall, and vacuole suggests an abundance of secretory mrnas.
the functional bias of the translationally variable genes towards 'abiotic stimuli’ suggests that the variation in translation state is likely a deliberate response of arabidopsis to subtle differences in growth conditions.
this was the case even though all four experiments were performed by the same person in the same growth chamber under nearly identical conditions.
given the diversity of stimuli listed, several different environmental factors may be involved.
this result is also in keeping with the notion that many mrnas that are functionally associated with environmental plasticity are regulated at the translational level  <cit> .
we suggest that noticeable effects on translation state can occur in response to very subtle  differences in growth conditions.
discussion
this study describes the first global analysis of defects in ribosome loading in a plant ribosomal protein mutant and one of few such studies in eukaryotes  <cit> .
the cytosolic rpl <dig>  a subunit of the large  subunit of the ribosome, is located near the 40s to 60s subunit interface on the lower hemisphere of the 60s, below the elongation factor binding site.
during translation elongation, the protein faces forward, toward the 3′ end of the mrna  <cit> .
a mutation in mouse rpl <dig>  causes a pleiotropic developmental phenotype.
the hypomorphic mouse mutant allele impairs rpl <dig> mrna splicing and synthesis of the rpl <dig> protein.
while the homozygous allele is lethal, heterozygotes have a reduced protein synthesis rate and altered rrna ribosome biogenesis  <cit> , while polysome profiles appear quite normal.
in contrast, a double mutant in the two rpl <dig> genes of saccharomyces cerevisiae is viable.
the yeast mutations reduce the growth rate in vivo and the protein synthesis rate in vitro, probably because of reduced p-site trna binding.
the rpl <dig> mutant yeast polysome profile shows stalled initiation complexes  <cit> .
rpl <dig> being one of the last proteins to be assembled into the 60s  <cit> , it is thought that other ribosomal proteins can join together successfully without rpl <dig>
the rpl24b mutation does not reveal translation elongation defects or ribosome biogenesis defects
one might consider that a mutation such as rpl24b affecting the large ribosomal subunit may cause a translation elongation defect.
such a defect might slow the movement of ribosomes and therefore raise ribosome numbers per mrna, and thus raise polysome loading, which would appear as an increase in translation state.
such a defect in the translation elongation cycle would be compounded in an exponential way by the length of the protein coding sequence.
we did not observe a consistent global increase in ribosome loading when we monitored the rna absorbance profile of our rpl24b mutant polysome gradients.
of note, we also did not detect evidence for half-mers, polysomes with a 40s subunit stalled on the start codon, as were observed in the yeast rpl <dig> mutant  <cit> .
third, the observed increase in ribosome loading in rpl24b was gene specific, and preferentially affected the r-protein mrnas, which are rather short, rather than long mrnas.
many ribosomal proteins play fundamental roles in ribosome assembly  <cit> .
however, we are not inclined to conclude that the rpl24b phenotype reflects a direct ribosome biogenesis defect, for the main reason that the fundamental aspects of the rpl24b mutant phenotype are mirrored by eif3h, a mutation in an initiation factor, which would not affect ribosome assembly directly.
taken together, we did not see clear hallmarks of defects in translation elongation or ribosome assembly in rpl24b, although we do not categorically rule out that they might exist.
the rpl24b mutant stimulated the expression or the ribosome loading of certain r-protein transcripts .
between the transcriptional and translational upregulation, a large proportion of r-proteins received some boost in expression in the rpl24b mutant.
a similar result was seen in eif3h <cit> .
it is conceivable that the mutant plants sense the defect in the translation apparatus, for example defective ribosomes lacking rpl <dig>  the molecular phenotype that we observe suggests the existence of a 'translation machinery surveillance’ response, which then stimulates expression of ribosomal proteins.
it is currently not possible to tease apart which components of the rpl24b mutant phenotype are due directly to the loss of the rpl24b protein and which are due to the indirect effects of the mutation on other r-proteins.
the problem of indirect effects is perhaps even more acute in vertebrates because ribosomal mutations trigger a p53-dependent nucleolar stress response pathway  <cit> .
whether ribosomal mutations in plants trigger an analogous response is currently unknown.
roles of rpl24b consistent with global measurements of translation
based on transient gene expression assays, arabidopsis rpl <dig> functions during translation reinitiation on the 35s rna of cauliflower mosaic virus   <cit> .
moreover, the rpl24b mutant was originally identified as shortvalve <dig> by virtue of its short valves, which are the walls of the seed pod  <cit> .
the rpl <dig> protein boosts the translation reinitiation of two uorf-containing auxin response transcription factor  mrnas, ettin  and monopteros   <cit> , in conjunction with eif3h  <cit> .
based on this information, we hypothesized that eif3h and rpl <dig> may be involved in similar initiation events or may coregulate the expression of uorf-containing or other mrnas.
the data obtained from the global analysis of ribosome loading only partly confirm this hypothesis.
indeed, several dozen mrnas depended on both eif3h and rpl24b for regular ribosome loading .
surprisingly, however, these mrnas were not highly enriched in uorfs.
vice versa, mrnas with uorfs that have clearly reduced ribosome loading in eif3h, tend to have a rather mild translation defect in rpl24b .
this result shows a certain discrepancy between translation as measured using gene expression assays and translation as measured using ribosome occupancy.
the result is actually reminiscent of earlier observations.
for example, the atbzip <dig> mrna, which is clearly eif3h dependent and rpl24b dependent in the gene expression assay  <cit> , has a relatively modest and statistically insignificant ribosome occupancy defect when measured by microarray  <cit> .
one probable cause is this; when the ribosome loading is reduced by twofold from six to three ribosomes per mrna, most mrna molecules will still be in a 'polysomal’ state.
one might further consider that the respective uorf-containing mrnas remain ribosome loaded in the rpl24b mutants because ribosomes persist on their uorf clusters.
we can also speculate that mutant rpl24b ribosomes might reinitiate translation imperfectly; they may translate portions of the major orf in an unproductive way that does not lead to a full protein.
for comparison, other mutations in the translation apparatus affect the fidelity of start codon recognition  <cit> ; aberrant initiation at non-aug start codons or non-canonical reading frames could potentially be detected using the ribosome footprinting technique  <cit> .
as we compare translation between rpl24b and eif3h, we should comment on the fact that the two experiments were performed under different light regimes.
the rpl24b experiment was performed under a 16 h light/8 h dark cycle and the plants were harvested approximately 4 h into the light period, while the other experiments were carried out in continuous light.
we do not think that the intermittent dark treatments contributed in a major way to the differences in translation.
by examining the mrna levels of characteristic clock gene transcripts we determined that the continuous-light-grown plants and the rpl24b plants were entrained in a similar way by the circadian clock.
in addition, when dark-treated plants are reilluminated, the ribosome loading for the majority of mrnas recovers within  <dig> minutes  <cit> .
even when fully etiolated seedlings are exposed to light, the major adjustment in polysome loading occurs within 4 h  <cit> .
the translational regulon of ribosomal proteins
this work suggests the presence of several translational regulons, defined as groups of mrnas that are subject to joint translational control across a variety of conditions.
we newly noticed that a subset of seed storage protein mrnas are jointly stimulated in their translation in the pab <dig> pab <dig> mutant.
since this group has not shown coregulation under other conditions, it does not qualify as a regulon as yet.
however, r-protein mrnas clearly form a regulon.
among the r-proteins, the organellar encoded ones are clearly set apart from the nuclear encoded ones.
given their correlated behavior in rpl24b and eif3h, the organellar r-protein mrnas appear to be part of a distinct regulon of mrnas.
the r-protein regulon holds steady under other experimental conditions, given that r-protein mrnas are among the most sensitive to hypoxia and other stresses  <cit> .
it is possible that certain r-proteins are excluded from the regulon.
however, aside from the 'incomplete’ r-protein mrnas, it is difficult to identify specific examples.
while certain r-proteins seemed to be stimulated less than others by the rpl24b and eif3h mutations, this could often be attributed to the fact that their translation state was already very high in the wild-type .
thus, it seems plausible that a large fraction of nuclear encoded r-protein mrnas are subject to coordinated translational regulation.
interestingly, while the response of the r-proteins to mutation is fairly uniform across the entire group, individual r-protein mrnas vary greatly in their ribosome occupancy .
this result has implications for ascribing functions to individual r-protein paralogs in the context of the hypothesis that the cell contains different ribosomes with specialized functions.
when two mutations in sister paralogs display different phenotypes, even though the patterns of transcript abundance of their mrnas are identical, such differences in phenotype may be due to different rates of translation.
although the r-protein regulon is functionally the best-characterized plant translational regulon, questions regarding the rna sequence features responsible for its regulation remain to be addressed in future work.
in general, it is unknown what causes the differences in ribosome occupancy among the paralogs.
given that their r-protein coding sequences are highly similar, one must consider whether ribosome occupancy is governed by sequences in the more variable 5′ and 3′ utrs.
CONCLUSIONS
a mutation affecting a large ribosomal subunit protein, rpl24b, stimulates ribosome loading in a similar way as a mutation in the eukaryotic initiation factor subunit, eif3h, whereas a mutation in poly-binding protein has a distinct, milder, spectrum of effects.
the most striking translational effects are centered on the ribosomal protein mrnas, suggesting that a large subset of these mrnas forms a regulon of translational control.

 the ribosome is responsible for the translation of all mrnas into protein. specific ribosomal protein mutations underlie certain human diseases. different ribosomal protein mutations also cause different spectra of developmental defects in metazoans and plants. these observations have raised interest in the contributions of the individual ribosomal proteins to translation of different mrnas. however, few studies have examined the consequences of ribosome defects on translation, and in the vast majority of case studies no genome-wide data are available. one exception concerns the ribosomal protein of the small subunit, rps a mutation in rps is responsible for cases of human diamond-blackfan anemia. the rps mutation causes both inhibition and stimulation of several dozen mrnas, including several that are implicated in the etiology of the disease. in another example, a mutation in mouse rpl results in a homeotic transformation affecting the ribcage. the rpl mutation interferes with translation of homeobox mrnas. genome-wide studies of translation under different stresses and environmental conditions have provided a wealth of information on global translation control in the model plant arabidopsis thaliana. however, even in arabidopsis, which has become one of the premier model organisms for translatome research, information on the role of specific translation factors is limited. the different isoforms of eukaryotic initiation factors eif4e and eif4g, which are components of the mrna cap-binding complex, contribute differentially to translation. the eif complex is the largest of the initiation factors. some evidence points toward a role for eif in promoting the translation of specific'client’ mrnas. only one global plant translation profile has been obtained from a mutant with a defect in the translation apparatus. in detail, mutations that delete the c-terminus of eif3h compromise translation reinitiation on mrnas containing upstream open reading frames, and also increase the polysome loading of mrnas with long 5′ leader or coding sequences. ribosomal protein rpl is a constituent of the cytosolic large 60s subunit. a mutant of a. thaliana in which one of three rpl24-encoding genes, rpl24b, is deleted, is characterized by developmental pattern defects in the base of the fruit. normal fruit patterning is regulated by uorfs and requires rpl24-dependent translation reinitiation on certain uorf-containing mrnas. the major rpl24b paralog, rpl24a, has also been implicated in the translation reinitiation on mrnas with multiple coding regions. in single-gene reporter gene expression assays of the uorf-containing mrna, atbzip the molecular defects in rpl24b resemble those in the eif3h mutant. the seedling phenotypes of eif3h and rpl24b, while not identical, overlap with respect to vascular development, gynoecium structures, and other organs. these data suggest that rpl24b and eif3h have related functions, although their exact biochemical roles are unclear. in arabidopsis, all ribosomal proteins are encoded by at least two paralogs, which are typically at least partially redundant. rpl24a and rpl24b are expressed at similarly high levels. the third and final paralog, rpl24c, is expressed at a fourfold lower level, and the protein may be enriched in the nucleolus. rpl has also been investigated in mouse and yeast. a mutation in the single mouse rpl gene, belly spot and tail, is homozygous lethal while heterozygotes show a pleiotropic developmental phenotype. for comparison, a double mutant in the two rpl genes of the yeast saccharomyces cerevisiae is not lethal. these results suggest that the yeast ribosome can assemble successfully without rpl in keeping with rpl being one of the last proteins to be assembled into the 60s subunit. another specific protein that has been implicated in plant translational control is poly-binding protein. by bridging between the 5′ cap binding complex and the 3′ poly tail, pabp is thought to enhance ribosome recycling and thus translation. in arabidopsis the class ii pabp genes are the more highly expressed in seedlings and encode the bulk of pabp in seedlings. there are no reports of mrna-specific translational defects for any pab mutant. for this study, we sought to identify the translational defects in two new types of mutants that affect the translation apparatus. we report here one of the first global analyses of ribosome occupancy in any organism for a ribosomal protein mutant, arabidopsis rpl24b. this mutant was chosen because of its functional association with the initiation factor eif3h, for which a mutant translatome is already available. we also report ribosome occupancy data in the arabidopsis pab pab double mutant. this mutant is viable, yet slightly dwarfed, indicative of compromised pabp activity; a pab pab pab triple mutant has not been recovered. the translational defects in the pab mutant were comparatively subtle and restricted to residual mrnas that are expressed highly in late embryogenesis. in contrast, the rpl24b mutation altered the ribosome occupancy of hundreds of mrnas. in keeping with the related developmental phenotypes of the rpl24b and eif3h mutations, their translational defects also overlapped. ribosomal protein mrnas were the most highly affected functional category. the ribosome occupancy of nuclear encoded r-proteins was typically stimulated by the rpl24b mutation. a large fraction of r-proteins, but not all, appear to belong to a single regulon of translational control.  identification of differentially translated genes in a ribosomal protein mutant seedlings of previously described mutant alleles were grown for the pab pab double mutant, the eif3h- mutant, and rpl24b. polysome microarray data from the rpl24b mutant and the pab pab mutant were collected using sucrose gradient fractionation of ribonucleoprotein complexes from arabidopsis seedlings and were processed as described in methods. in parallel, equivalent data from a published experiment on the eif3h mutant were reanalyzed from the hybridization signals using the same procedure as for rpl24b. the translation state is defined as the ratio between the mrna signal in the polysomal: non-polysomal fractions and is displayed as a log value. for example, an mrna that is 80% polysomal and 20% non-polysomal receives a tl of and a log tl of +. for this reason the tl value is completely independent of the transcript level. we carefully evaluated many different data processing procedures in order to optimize detection of differentially translated genes. dtgs between mutant and wild-type were first mined using a stringent false-discovery-rate cut-off of 5%. from the entire set of probe sets, we identified and dtgs for rpl24b and eif3h, respectively. because this number for eif3h was small compared to the number of dtgs identified in the earlier publication, we sought to expand the gene set. using the additional prefiltering methods laid out in the methods, we assembled a non-redundant set of dtgs, for rpl24b and for eif3h. comparison of translation defects between rpl24b and eif3h because translation of atbzip auxin response factor and arf depend on functional eif3h and rpl24b, one might hypothesize that eif3h and rpl24b control the ribosome loading of identical sets of client mrnas. we tested this hypothesis by comparing clusters of genes that are altered at the translation level in eif3h and rpl24b mutants. among the dtgs in rpl24b or eif3h, the direction and the degree of mistranslation were significantly correlated. this indicates that translation of many mrnas are coregulated by, or codependent on, rpl24b and eif3h. remarkably, among all the mrnas that were overtranslated in eif3h, the vast majority had a trend towards overtranslation in the rpl24b dataset; the same held true for undertranslated mrnas. this result indicates that a substantial and significant number of arabidopsis mrnas are codependent on eif3h and rpl24b for appropriate translation. the mrnas that were translationally stimulated in both rpl24b and eif3h mutants were enriched in ribosomal protein mrnas while the undertranslated ones had no detectable functional bias. the effects of rpl24b and eif3h on transcript abundance were also correlated. because rpl and eif3h are translation factors, we presume that most of the changes at the transcript level are a consequence, rather than a cause, of alterations in translation. we also note that the translational defects in the rpl24b mutant are not strongly correlated with changes in total transcript levels, similar as for eif3h. to work out whether there are mrnas that depend on only of the genes, rpl24b or eif3h, but not on the other, we performed fuzzy k-means clustering on a total of dtgs. two of six clusters, clusters and corresponded to the strongly coregulated mrnas. two other clusters, clusters and showed more limited coregulation, that is, a strong response in one mutant and a moderate trend in the other. the last two clusters, clusters and contained mrnas whose response was much stronger in eif3h than in rpl24b. there were few rpl24b-specific mrnas. overall, these results underscore the significant overlap between sets of mrnas mistranslated in eif3h and rpl24b mutants. these new findings bolster the conclusion that rpl24b and eif3h play related roles in eukaryotic gene expression. translation states in the pab pab double mutant the poly binding proteins are widely thought to stimulate translation in a cellular context by helping to juxtapose 3′ end and 5′ end of mrnas and by assisting with ribosome recycling. pab pab and pab are the three major pabp genes expressed in seedlings and are functionally redundant. the pab pab double mutant is mildly dwarfed. we examined the ribosome occupancy of mrnas in the pab pab double mutant seedlings. overall, the pab pab double mutant had few gene-specific alterations in its ribosome occupancy. the primary fdr-controlled alteration was an approximately twofold increase in ribosome occupancy for a small group of seed storage protein mrnas. this result is of interest, as seed storage protein mrnas are under translational control in various other plants. less stringent filtering using an alternative ad hoc method to identify dtgs recovered oleosins, which are additional seed storage mrnas, and late embryogenesis abundant mrnas among the mrnas with increased ribosome loading in pab pab it is notable that pab the major remaining pabp mrna in the pab pab mutant, was also translationally stimulated. this result favors the hypothesis that pabp mrnas are subject to negative translational autoregulation via poly stretches in their 5′ untranslated regions. we compared the translational alterations in the rpl24b mutant with those in pab pab not even a weak correlation was detected. this important result suggests that different perturbations of the translational machinery affect different sets of mrnas. because most alterations in ribosome occupancy in pab pab were small and statistically insignificant on a single-gene basis, we searched for global patterns. short mrnas were enriched among the mrnas that were translationally inhibited in the pab pab mutant. this trend could be attributed to the length of the coding sequence as well as the length of the 5′ utr, while the length of the 3′ utr contributed little if at all. analysis of mrna transcript levels in the pab pab mutant revealed and significantly downregulated and upregulated genes at the mrna transcript level. the transcript levels of pab and pab were reduced, as expected given the t-dna insertions in these genes in the pab pab strain. interestingly, among the mrnas with >2-fold increased ribosome occupancy in pab pab all except are late-embryogenesis mrnas whose level declines up to 1000-fold during seed germination, the developmental period preceding our analysis. of them, were also upregulated at the transcript level in pab pab for pab pab and in contrast to the situation in rpl24b, the correlation between translational and transcript-level stimulation continued among the less strongly stimulated mrnas. this result suggests that wild-type pabp might couple transcript turnover and translation state for these late embryogenesis mrnas. overtranslation of ribosomal protein mrnas in rpl24b and eif3h we performed genome ontology analyses of the six clusters of rpl24b or eif3h dependent mrnas identified by fuzzy k-means clustering. clusters and were both highly enriched for r-protein mrnas. of note, cytosolic r-protein mrnas are coordinately affected in response to sucrose starvation, drought, hypoxia stress, and unanticipated darkness. however, in each of these stresses, the r-protein mrnas are undertranslated rather than overtranslated. to detect smaller effects of rpl24b and eif3h on the ribosomal protein mrnas, we adopted the ad hoc filtering method to identify differentially translated genes. the ad hoc method does not control for fdr. this method enriched even more significantly for r-proteins. of all the cytosolic r-protein mrnas for which the arrays yielded data, 41% were found in clusters to a -fold enrichment. this result suggests that the ad hoc method, which is less conservative than the fdr method, produces fewer false negative calls. these results, therefore, confirm in more detail that many but not all cytosolic r-protein mrnas depend on rpl24b and eif3h at the translation level and could define a regulon of translational control. mrnas that were affected by eif3h or rpl24b mutations were not generally perturbed by the pab pab mutations or by a mutation in the k subunit of eif. we next visualized the r-protein cohort at a gene-specific level. among mrnas encoding cytosolic r-proteins the pattern of overtranslation in the rpl24b mutant was generally mirrored and enhanced in the eif3h mutant. in contrast, the pattern in the pab pab mutant bore no resemblance to that in rpl24b or eif3h. the joint overtranslation in rpl24b and eif3h did not generally extend to the translation initiation factors; among the few that were jointly stimulated, we found a few eif subunits. the mutation in rpl24b also stimulated the mrna transcript levels of many cytosolic r-protein mrnas, but the changes at the transcript level were not obviously correlated with those at the translation level. among the mrnas for organellar r-proteins, those encoded in the nucleus were translationally stimulated in a similar fashion in the two mutants, although the trend was weaker than for the nuclear-encoded cytosolic proteins. in contrast, mrnas for r-proteins that are encoded in the organelles themselves, that is, the plastid or mitochondrion, all had reduced ribosome loading in the rpl24b and eif3h mutants. the effect on the organellar mrnas must be indirect because rpl and eif3h function only in the cytosol. it should be noted that our array data are biased for polyadenylated mrnas. in the organelles, polyadenylation is a precursor for degradation. thus, our results suggest that, in the wild-type chloroplasts, polyadenylated mrnas remain substantially ribosome loaded, whereas in the mutants, the ribosome loading of the polyadenylated mrnas is reduced. fine structure analysis of the translation status of ribosomal protein mrnas because the r-protein mrnas were reliably expressed and showed robust cotranslation among themselves and between rpl24b and eif3h mutants, we characterized their translational coregulation in greater detail. figure 5b shows that the ribosome occupancy of the r-protein mrnas in wild-type plants varied dramatically among the different mrnas. certain extremely short mrnas should have low ribosome occupancy, simply because few ribosomes fit onto the coding sequence of a small mrna. this was indeed the case for several of the smallest r-proteins. however, even among the paralogs of one family, it was common for one paralog to be highly translated, and another paralog to be poorly translated. however, the reproducibility of the ribosome occupancy between the two replicate experiments was excellent. certain r-protein mrnas have incomplete coding sequences and have been marked as pseudogenes. these mrnas tended to have lower ribosome occupancy, additional file figure 5a shows how ribosome occupancy of the r-protein mrnas was affected in the rpl24b and eif3h mutants. the following numbers of mrnas cleared statistical significance at fdr <: mrnas that changed ribosome loading in eif3h and in rpl24b. several generalizations are supported by these data. first, certain mrnas with very high ribosome occupancy in the wild-type did not increase their tl in the mutant, presumably simply because tl was already close to maximal. vice versa, mrnas with medium or low ribosome occupancy in the wild-type had an upward trend in the mutant. second, for many r-protein families, the degree of translational dependence differed between the different paralogs; clear examples include l l l l18a, l l35a, p sa, s s s15a, s and s third, in keeping with the pattern of cotranslation between rpl24b and eif3h described earlier the paralog with the strongest stimulation in rpl24b often had the strongest stimulation in eif3h. for examples, see l l l l l l18a, l l l l p s s s and s clear exceptions were rare. rpl presents one of the few exceptions, almost certainly because the rpl24b mrna is truncated by a deletion in the rpl24b mutant. fourth, in a minority of r-protein families all paralogs were slightly reduced in their ribosome loading in the rpl24b mutant. most of these r-proteins were small proteins of less than to 13 kda. examples are l, l35a, l36a, l37a, l, s, s, s, s, and s. in contrast, most of the larger r-proteins had at least one paralog that was translationally stimulated in the rpl24b mutant. on this topic, we note that the translation of these short mrnas was generally normal in the eif3h mutant. in the eif3h mutant, long mrnas are preferentially inhibited in their ribosome loading, but short mrnas are not. this is one exception from the general rule of correlated translation behavior between the two mutants. finally, certain r-protein paralogs code for incomplete open reading frames. of the eight such cases for which we have data, none were translationally stimulated in either of the mutants, in sharp contrast to the general pattern. indeed, the two mrnas with the strongest translational repression in eif3h, rps12b and rps25c, both encode incomplete orfs and are annotated as pseudogenes in the arabidopsis information resource, release it is unclear why these mrnas are more sensitive to the mutations. taken together, considering the detailed annotation features just discussed, it appears that the r-protein regulon probably includes more mrnas than was evident from figure  while figure  suggested that certain r-protein mrnas might be excluded from the regulon, detailed inspection of figure  makes this hypothesis harder to sustain. of the cytosolic r-proteins above 13 kda, the majority showed translational stimulation in both rpl24b and eif3h for at least one paralog. paralogs that were stimulated less in the mutants often had a near maximal translation state in the wild-type. these mrnas should be counted as legitimate members of the r-protein regulon. other paralogs that bucked the general pattern and should not be considered part of the r-protein regulon code for incomplete r-proteins and rpl24b itself. molecular features of undertranslated and overtranslated mrnas given that the rpl24b mutant shared a common set of translationally stimulated and translationally inhibited mrnas with eif3h, we examined whether these mrnas harbored the same features that render some arabidopsis mrnas eif3h-dependent. these features are uorfs and a long coding region. as shown in additional file 6: figure s uorfs only played a weak role in causing poor translation in rpl24b. in detail, a statistically significant association between uorfs and rpl24b-dependent translation was seen only within the middle range of the distribution, but not for the more strongly rpl24b-dependent mrnas. the association of orf length with rpl24b-dependence was also weak. in contrast, for the eif3h dataset, our reanalysis of the earlier data reproduced the conclusion that uorfs correlate with eif3h dependence. to get a clearer view of this unexpected result, we examined the occurrence of uorfs among the undertranslated genes found in clusters and. the mrnas that were clearly codependent on both rpl24b and eif3h, were not enriched for uorfs, while the mrnas that were strongly eif3h-dependent but weakly rpl24b-dependent tended to be the ones with uorfs. apparently, the rpl24b mutation caused only a minor deficiency in ribosome loading for the uorf-containing mrnas. this was surprising, because if one measures reporter protein translation downstream from a uorf in the rpl24b mutant, the deficiency in translation can be quite pronounced. in summary, the effects of the mutations in eif3h and rpl24b on ribosome occupancy are related but not identical. stochastic variation of mrna translation states in wild-type plants data on mrna translation states can be variable between biological replicates. we examined the structure of the variation in translation states that we observed among different rna samples from wild-type seedlings, collected from seedlings grown on defined agar medium under largely identical experimental conditions. we focused on the 20% of mrnas with the largest variation in translation state among the replicates, that is, a standard deviation above. likewise, we identified a group of mrnas with highly variable mrna transcript levels. gene ontology of the translationally variable mrnas revealed that the most enriched biological process was'response to abiotic stimulus’. within this category, many different stimuli and stresses were represented and none stood out. the most enriched cellular compartment was the'chloroplast’, and multiple compartments such as'stroma’ and'envelope’, but not'thylakoid’, were overrepresented. the overrepresentation of plasma membrane, apoplast, cell wall, and vacuole suggests an abundance of secretory mrnas. the functional bias of the translationally variable genes towards'abiotic stimuli’ suggests that the variation in translation state is likely a deliberate response of arabidopsis to subtle differences in growth conditions. this was the case even though all four experiments were performed by the same person in the same growth chamber under nearly identical conditions. given the diversity of stimuli listed, several different environmental factors may be involved. this result is also in keeping with the notion that many mrnas that are functionally associated with environmental plasticity are regulated at the translational level. we suggest that noticeable effects on translation state can occur in response to very subtle differences in growth conditions. discussion this study describes the first global analysis of defects in ribosome loading in a plant ribosomal protein mutant and one of few such studies in eukaryotes. the cytosolic rpl a subunit of the large subunit of the ribosome, is located near the 40s to 60s subunit interface on the lower hemisphere of the 60s, below the elongation factor binding site. during translation elongation, the protein faces forward, toward the 3′ end of the mrna. a mutation in mouse rpl causes a pleiotropic developmental phenotype. the hypomorphic mouse mutant allele impairs rpl mrna splicing and synthesis of the rpl protein. while the homozygous allele is lethal, heterozygotes have a reduced protein synthesis rate and altered rrna ribosome biogenesis, while polysome profiles appear quite normal. in contrast, a double mutant in the two rpl genes of saccharomyces cerevisiae is viable. the yeast mutations reduce the growth rate in vivo and the protein synthesis rate in vitro, probably because of reduced p-site trna binding. the rpl mutant yeast polysome profile shows stalled initiation complexes. rpl being one of the last proteins to be assembled into the 60s, it is thought that other ribosomal proteins can join together successfully without rpl the rpl24b mutation does not reveal translation elongation defects or ribosome biogenesis defects one might consider that a mutation such as rpl24b affecting the large ribosomal subunit may cause a translation elongation defect. such a defect might slow the movement of ribosomes and therefore raise ribosome numbers per mrna, and thus raise polysome loading, which would appear as an increase in translation state. such a defect in the translation elongation cycle would be compounded in an exponential way by the length of the protein coding sequence. we did not observe a consistent global increase in ribosome loading when we monitored the rna absorbance profile of our rpl24b mutant polysome gradients. of note, we also did not detect evidence for half-mers, polysomes with a 40s subunit stalled on the start codon, as were observed in the yeast rpl mutant. third, the observed increase in ribosome loading in rpl24b was gene specific, and preferentially affected the r-protein mrnas, which are rather short, rather than long mrnas. many ribosomal proteins play fundamental roles in ribosome assembly. however, we are not inclined to conclude that the rpl24b phenotype reflects a direct ribosome biogenesis defect, for the main reason that the fundamental aspects of the rpl24b mutant phenotype are mirrored by eif3h, a mutation in an initiation factor, which would not affect ribosome assembly directly. taken together, we did not see clear hallmarks of defects in translation elongation or ribosome assembly in rpl24b, although we do not categorically rule out that they might exist. the rpl24b mutant stimulated the expression or the ribosome loading of certain r-protein transcripts. between the transcriptional and translational upregulation, a large proportion of r-proteins received some boost in expression in the rpl24b mutant. a similar result was seen in eif3h. it is conceivable that the mutant plants sense the defect in the translation apparatus, for example defective ribosomes lacking rpl the molecular phenotype that we observe suggests the existence of a'translation machinery surveillance’ response, which then stimulates expression of ribosomal proteins. it is currently not possible to tease apart which components of the rpl24b mutant phenotype are due directly to the loss of the rpl24b protein and which are due to the indirect effects of the mutation on other r-proteins. the problem of indirect effects is perhaps even more acute in vertebrates because ribosomal mutations trigger a p53-dependent nucleolar stress response pathway. whether ribosomal mutations in plants trigger an analogous response is currently unknown. roles of rpl24b consistent with global measurements of translation based on transient gene expression assays, arabidopsis rpl functions during translation reinitiation on the 35s rna of cauliflower mosaic virus. moreover, the rpl24b mutant was originally identified as shortvalve by virtue of its short valves, which are the walls of the seed pod. the rpl protein boosts the translation reinitiation of two uorf-containing auxin response transcription factor mrnas, ettin and monopteros, in conjunction with eif3h. based on this information, we hypothesized that eif3h and rpl may be involved in similar initiation events or may coregulate the expression of uorf-containing or other mrnas. the data obtained from the global analysis of ribosome loading only partly confirm this hypothesis. indeed, several dozen mrnas depended on both eif3h and rpl24b for regular ribosome loading. surprisingly, however, these mrnas were not highly enriched in uorfs. vice versa, mrnas with uorfs that have clearly reduced ribosome loading in eif3h, tend to have a rather mild translation defect in rpl24b. this result shows a certain discrepancy between translation as measured using gene expression assays and translation as measured using ribosome occupancy. the result is actually reminiscent of earlier observations. for example, the atbzip mrna, which is clearly eif3h dependent and rpl24b dependent in the gene expression assay, has a relatively modest and statistically insignificant ribosome occupancy defect when measured by microarray. one probable cause is this; when the ribosome loading is reduced by twofold from six to three ribosomes per mrna, most mrna molecules will still be in a'polysomal’ state. one might further consider that the respective uorf-containing mrnas remain ribosome loaded in the rpl24b mutants because ribosomes persist on their uorf clusters. we can also speculate that mutant rpl24b ribosomes might reinitiate translation imperfectly; they may translate portions of the major orf in an unproductive way that does not lead to a full protein. for comparison, other mutations in the translation apparatus affect the fidelity of start codon recognition; aberrant initiation at non-aug start codons or non-canonical reading frames could potentially be detected using the ribosome footprinting technique. as we compare translation between rpl24b and eif3h, we should comment on the fact that the two experiments were performed under different light regimes. the rpl24b experiment was performed under a 16 h light/8 h dark cycle and the plants were harvested approximately 4 h into the light period, while the other experiments were carried out in continuous light. we do not think that the intermittent dark treatments contributed in a major way to the differences in translation. by examining the mrna levels of characteristic clock gene transcripts we determined that the continuous-light-grown plants and the rpl24b plants were entrained in a similar way by the circadian clock. in addition, when dark-treated plants are reilluminated, the ribosome loading for the majority of mrnas recovers within minutes. even when fully etiolated seedlings are exposed to light, the major adjustment in polysome loading occurs within 4 h. the translational regulon of ribosomal proteins this work suggests the presence of several translational regulons, defined as groups of mrnas that are subject to joint translational control across a variety of conditions. we newly noticed that a subset of seed storage protein mrnas are jointly stimulated in their translation in the pab pab mutant. since this group has not shown coregulation under other conditions, it does not qualify as a regulon as yet. however, r-protein mrnas clearly form a regulon. among the r-proteins, the organellar encoded ones are clearly set apart from the nuclear encoded ones. given their correlated behavior in rpl24b and eif3h, the organellar r-protein mrnas appear to be part of a distinct regulon of mrnas. the r-protein regulon holds steady under other experimental conditions, given that r-protein mrnas are among the most sensitive to hypoxia and other stresses. it is possible that certain r-proteins are excluded from the regulon. however, aside from the'incomplete’ r-protein mrnas, it is difficult to identify specific examples. while certain r-proteins seemed to be stimulated less than others by the rpl24b and eif3h mutations, this could often be attributed to the fact that their translation state was already very high in the wild-type. thus, it seems plausible that a large fraction of nuclear encoded r-protein mrnas are subject to coordinated translational regulation. interestingly, while the response of the r-proteins to mutation is fairly uniform across the entire group, individual r-protein mrnas vary greatly in their ribosome occupancy. this result has implications for ascribing functions to individual r-protein paralogs in the context of the hypothesis that the cell contains different ribosomes with specialized functions. when two mutations in sister paralogs display different phenotypes, even though the patterns of transcript abundance of their mrnas are identical, such differences in phenotype may be due to different rates of translation. although the r-protein regulon is functionally the best-characterized plant translational regulon, questions regarding the rna sequence features responsible for its regulation remain to be addressed in future work. in general, it is unknown what causes the differences in ribosome occupancy among the paralogs. given that their r-protein coding sequences are highly similar, one must consider whether ribosome occupancy is governed by sequences in the more variable 5′ and 3′ utrs.  a mutation affecting a large ribosomal subunit protein, rpl24b, stimulates ribosome loading in a similar way as a mutation in the eukaryotic initiation factor subunit, eif3h, whereas a mutation in poly-binding protein has a distinct, milder, spectrum of effects. the most striking translational effects are centered on the ribosomal protein mrnas, suggesting that a large subset of these mrnas forms a regulon of translational control. 
to identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mrnas to examine the defects in translation in a poly binding protein mutant, pab pab as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve these data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. genome-wide assays performed in arabidopsis and other organisms have revealed that the translation status of mrnas responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. the mutation of rpl24b stimulated the ribosome occupancy of mrnas for nuclear encoded ribosomal proteins. third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. many of these mrnas were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mrnas. first, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. some sister paralogs also differed in their response to the rpl24b mutation. in contrast to rpl24b, the pab pab mutant revealed few gene specific translational defects, but a group of seed storage protein mrnas were stimulated in their ribosome occupancy. in the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected biological replicates of translation states from wild-type seedlings. detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mrnas. the data also shed light on the fine structure of the regulon of ribosomal protein mrnas. rpl and eif3h play similar but non-identical roles in eukaryotic translation. we defined 20% of mrnas as having a high variance in their translation state.
 genome-wide assays performed in arabidopsis and other organisms have revealed that the translation status of mrnas responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. to identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mrnas to examine the defects in translation in a poly binding protein mutant, pab pab as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve  the mutation of rpl24b stimulated the ribosome occupancy of mrnas for nuclear encoded ribosomal proteins. detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mrnas. first, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. second, a group of mrnas with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on rpl24b differed from regular mrnas. third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. some sister paralogs also differed in their response to the rpl24b mutation. in contrast to rpl24b, the pab pab mutant revealed few gene specific translational defects, but a group of seed storage protein mrnas were stimulated in their ribosome occupancy. in the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected biological replicates of translation states from wild-type seedlings. we defined 20% of mrnas as having a high variance in their translation state. many of these mrnas were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mrnas.  these data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. rpl and eif3h play similar but non-identical roles in eukaryotic translation. the data also shed light on the fine structure of the regulon of ribosomal protein mrnas. translation stateribosome occupancyrpl24pabpregulonarabidopsis 