BACKGROUND
post-translational modification of proteins is a typical mark of signal transduction pathways through which organisms are able to react quickly to changes in their surrounding by expanding the structural and functional diversity of the proteome.
arginine methylation is a common post- translational modification in eukaryotic organisms, and is catalyzed by a family of enzymes known as protein arginine methyltransferases .
this post-translational modification modulates a myriad of cellular processes through its effects on proteins involved in the regulation of chromatin structure, transcription, rna processing, signal transduction and cellular differentiation, among other processes  <cit> .
prmts are classified in four groups: type i prmts that generate ω-ng-monomethyl arginine  and ω-ng,ng-asymmetric dimethylarginines , type ii prmts that generate ω-ng-monomethyl arginine and ω-ng,ng-symmetric dimethylarginines , type iii prmts that generate only ω-ng-monomethyl arginine, and type iv that generate only δ-ng-monomethyl arginine  <cit> .
currently, four different genes encoding prmts have been described in saccharomyces cerevisiae, eight in oryza sativa, and nine in humans, drosophila and arabidopsis thaliana  <cit> .
in mammals, there are six well characterized arginine methyltransferases, five of them corresponding to type i prmts ; while the only type ii prmt known so far is prmt <dig>  it has been proposed that there is no major redundancy between these enzymes, at least in mammals, since knock-out mice for each prmt display clearly different phenotypes  <cit> .
it is known that proteins that possess glycine and arginine- rich  motifs are often targets of prmts.
transcriptional regulation by methylation of histones and non-histone proteins is one of the more characterized functions of prmts.
it has been reported that prmt <dig> methylates arg  <dig> of histone h <dig>  prmt <dig> methylates arg  <dig>  arg  <dig> and arg  <dig> of histone h <dig> and that prmt <dig> methylates arg  <dig> of histone h <dig> and arg  <dig> of histone h <dig>  in mammals, formation of adma in histones by prmt <dig> and prmt <dig> participates in gene activation while formation of sdma by prmt <dig> is associated with gene repression  <cit> .
arginine methylation has also been associated with the regulation of the initiation and elongation steps of transcription.
the recruitment of prmt <dig> to transcriptional promoters results in methylation of histone acetyltransferases resulting in a positive effect on transcription  <cit> , while prmt <dig> methylates the transcriptional elongation factor spt <dig> regulating its interaction with rna polymerase ii  <cit> .
another well characterized role of prmts is the regulation of rna processing.
rna binding proteins  fulfill numerous tasks ensuring the proper processing and folding as well as the stabilization and localization of rnas and mrna translation.
these proteins represent major targets for prmts because most hnrnps possess gar motifs.
prmts are also known to methylate sm spliceosomal proteins b, b’, d <dig> and d <dig>  and this mediates their assembly into mature small nuclear ribonucleoprotein particles , and has been associated with a role for prmt <dig> in the regulation of pre-mrna splicing  <cit> .
on the other hand, prmt <dig> regulates the coupling of transcription and mrna processing through methylation of splicing factors  <cit> .
strikingly, only a few studies have compared side by side the roles of distinct prmts in the regulation of different physiological and molecular processes.
in fact, several of these studies showed that prmt <dig> and prmt <dig> simultaneously control myogenesis, with both proteins having similar positive roles in the control of expression of genes known to play a key role regulating this developmental process  <cit> .
this is at odds with the current view that assumes that prmt <dig>  a type i prmt, acts as a co-activator of gene expression, while prmt <dig>  the main type ii prmt, acts as a transcriptional repressor  <cit> .
whether the similar positive role that prmt <dig> and prmt <dig> play in the control of myogenesis is the exception or the rule is not known.
discriminating between these two alternatives requires a side-by-side comparison of the effects of prmt <dig> and prmt <dig> on gene expression at a genome-wide level.
interestingly, both prmt <dig> and prmt <dig> have been proposed to play key roles in the regulation of pre-mrna splicing.
as previously reviewed, this regulatory role may be exercised through direct methylation of core spliceosomal proteins or, alternatively or in addition, through regulation of the coupling between transcription and mrna processing  <cit> .
however, while genome-wide analyses of pre-mrna splicing have been conducted to characterize prmt <dig> deficient plants or animals, similar global analysis are missing for mutants affected in prmt <dig>
plants are ideal organisms to conduct a side-by-side comparison of the roles of prmt <dig> and prmt <dig> on gene expression and rna processing, because mutant plants lacking these proteins are viable and fertile, while defects in these two genes are lethal in mammals.
in arabidopsis thaliana there are seven type i arginine methyltransferases  and, as in mammals, one type ii enzyme .
to date, the most intensively studied prmt in plants is prmt <dig>  which controls flowering time, circadian rhythms photomorphogenic development and salt tolerance acting on epigenetic regulation of gene expression and on pre-mrna splicing of a sub-set of genes  <cit> .
regarding type i prmts, it has been shown that prmt <dig>  prmt1b, and prmt4a together with prmt4b, are all involved in the regulation of flowering time  <cit> .
both type i and type ii prmts appear to regulate flowering time in arabidopsis thaliana through effects on flc expression.
flc is a mads box transcription factor that represses flowering, and prmt <dig>  prmt <dig> mutants and the prmt4a;4b double mutant all exhibit increased flc expression, which is partially responsible for the delayed flowering observed in these mutant plants  <cit> .
so far, the only phenotype that has been reported for prmt4a;4b mutant plants is delayed flowering  <cit> .
interestingly, this mutant resembles prmt <dig> plants at the morphological level, showing some degree of growth retardation and dark green leaves.
however, whether prmt4a;4b also exhibits other physiological alterations present in prmt <dig> mutants such as defects in circadian clock function, photomorphogenic development or salt stress tolerance is not known.
in this study we compared side by side the role of prmt <dig> and prmt4s in the regulation of several physiological processes in arabidopsis thaliana, and coupled this with a genome-wide comparison of their effects on gene expression and pre-mrna splicing using rna-seq.
we found that prmt <dig> and prmt4a;4b mutants not only displayed similar alterations in flowering time regulation, but also exhibited reduced inhibition of hypocotyl elongation under both red and blue light, revealing defects in light signaling.
in addition, similarly to what has been reported for prmt <dig> mutants, we found that prmt4a;4b double mutants exhibited reduced tolerance to salt stress.
however, in contrast to what is observed in prmt <dig> mutants, the prmt4a;prmt4b double mutant did not exhibit alterations in circadian rhythms.
rna-seq data showed that both mutants display similar alterations in the expression of genes related to: transcription, mrna processing, mrna splicing, translation, light signaling, response to hormones and both abiotic and biotic stress.
in addition, we also found that these mutants have similar alterations in alternative splicing  of genes related to translation, light signaling, response to hormones and both abiotic and biotic stress.
finally, we observed a significant number of novel intron retention events in both mutants, revealing alterations in a subset of constitutive as well as as events in those plants.
hence, this study shows that in arabidopsis thaliana, type i prmt4s and type ii prmt <dig> regulate overlapping as well as distinct physiological processes, most likely through similar effects on gene expression and pre-mrna splicing of a subset of key regulatory genes.
additionally, this study is the first to report an analysis of the role of prmt4s on pre-mrna splicing at a global level.
RESULTS
prmt <dig> and prmt <dig> control overlapping as well as distinct physiological processes
before conducting a genome-wide comparison of the roles of prmt <dig> and prmt <dig> in the regulation of gene expression and rna processing in arabidopsis thaliana, we performed a side by side analysis of the effect of these genes on the control of several developmental and physiological processes.
in particular, we focused the analysis on clock-associated processes, such as flowering time regulation, photomorphogenic responses and circadian rhythms, as well as on salt stress tolerance.
all these physiological and developmental processes have previously been shown to be affected in prmt <dig> mutants but have not been characterized in prmt4a;4b mutants, with the exception of flowering time.
indeed, as previously reported individually for prmt <dig> and prmt4a;4b,  <cit> , both mutants showed a late flowering phenotype compared to wild-type plants of the col- <dig> accession.
however, while both mutants displayed a clear and similar late flowering phenotype under short day photoperiods, the late flowering phenotype of prmt <dig> was much stronger than that of prmt4a;4b mutant plants under long day photoperiods .figure  <dig>
side by side comparison of the physiological roles of prmt <dig> and prmt <dig>   flowering time measured as the number of rosette leaves at bolting in long day  and short day  photoperiods.
anova followed by a tukey’s multiple comparision test was used to evaluate the statistical significance of the differences observed between genotypes.
errors bars indicate sd .
circadian rhythms of leaf movement in continuous light .
period of the circadian rhythms was estimated with brass  <dig>  software .
hypocotyl length of seedlings grown under continuous red light  or continuous blue light  .
comparison of root growth on ms medium with 100 mm nacl.
root growth was measured relative to controls.
more than  <dig> roots were measured for each data point.
data represents the mean with sd of three independent experiments .
salt tolerance was assessed through the analysis of survival rate on ms medium containing 160 mm nacl.
data represents the mean with sd of three independent experiments .
we and others have previously shown that prmt <dig> plays a key role in the regulation of circadian rhythms in arabidopsis  <cit> , but whether prmt <dig> contributes to the regulation of clock function is not known.
to evaluate this we monitored circadian rhythms in leaf movements in wild-type plants, prmt <dig> and prmt4a;4b mutant plants.
interestingly, while prmt <dig> mutant plants showed the previously described long period phenotype for leaf movement, the rhythms observed in prmt4a;4b mutants were similar to those of wild-type plants .
in order to analyze the role of these prmts in photomorphogenesis we evaluated light inhibition of hypocotyl elongation during de-etiolation in seedlings exposed to different fluence rates of red and blue light.
we found that both prmt <dig> and prmt4a;4b mutants were hyposensitive to red light at all fluence rates tested .
a similar phenotype was observed for both mutants under blue light as well .
prmt <dig> has been shown to regulate salt stress tolerance in arabidopsis  <cit> .
to test if prmt4a, together with prmt4b, were also involved in the regulation of this physiological response, we analyzed root growth, a process affected by high salt concentrations.
root length of wild-type plants grown on ms medium containing 100 mm nacl was approximately 80% of that shown by wild-type plants grown on ms medium alone.
in contrast, root length of prmt <dig> and prmt4a;4b mutants grown on medium containing the same concentration of salt was only 30% and 50%, respectively, relative to that of plants from these genotypes grown on ms medium .
we also conducted a survival rate assay and found that growth of both prmt <dig> and prmt4a;4b mutants was almost completely inhibited in medium containing 160 mm nacl, while wild-type plants displayed only a slightly inhibited growth rate under this condition .
altogether this shows that prmt <dig> and, to a lesser extent prmt4a, together with prmt4b, are all involved in the control of salt stress tolerance in arabidopsis.
impact of prmt <dig> and prmt4s on genome wide gene expression
in order to study the extent of the regulatory impact of prmt <dig> and prmt4s on gene expression we analyzed the transcriptome of wild-type, prmt <dig>  and prmt4a;b plants grown under standard non-stressful conditions  using rna-seq.
we found  <dig> genes over-expressed and  <dig> under-expressed in prmt <dig> mutants, as well as  <dig> genes over-expressed and  <dig> under-expressed in prmt4a;4b mutants, relative to wild-type plants.
strikingly, many of the differentially expressed genes were similarly affected in both mutants, with  <dig> and  <dig> genes over-expressed or under-expressed in common, respectively.
on the other hand, only  <dig> genes, out of a total of  <dig> and  <dig> genes differentially expressed in either prmt <dig> or prmt4a;b, respectively, were antagonistically affected in the mutants.
therefore, it is highly unlikely that the prmts analyzed here exhibit opposite biological and molecular roles .figure  <dig>
impact of prmt <dig> and prmt <dig> on genome wide gene expression.
overlap between differentially expressed genes in both prmt <dig> and prmt4a;4b mutants .
representation factor of genes co- regulated by prmt <dig> and prmt4s in the selected functional categories.
*: indicates over-representation compared to random.
significance was assessed using a hypergeometric test .
log <dig> fold change of representative genes co- regulated by prmt <dig> and prmt4s .
the differentially expressed genes that were similarly affected in both mutants were categorized into functional groups based on gene ontology.
fifteen functional categories of our interest were examined in detail determining the representation factor for each category.
the representation factor is the number of overlapping genes observed divided by the expected number of overlapping genes drawn randomly from two independent groups.
among the up-regulated genes, we found a significant enrichment  for categories corresponding to transcription, mrna processing, rna splicing, response to light, response to hormones, abiotic stress and biotic stress.
for down-regulated genes, we found significant enrichment for genes associated with primary metabolism, translation, response to light, abiotic stress and biotic stress .
none of the categories studied here displayed a statistically significant under-representation .
finally, we also found a number of genes that were significantly affected only in prmt <dig> or prmt4a;b mutants, some of which may be responsible for the partially distinct phenotypes of the mutants, such as the differential effect on circadian rhythmicity .
altogether, our physiological and molecular analysis suggests that these prmts regulate most of the mentioned processes controlling the expression of a common set of genes.
indeed, when we analyzed in detail the identity of the co-regulated genes associated with the biological processes mentioned above, we found that many of them corresponded to transcription factors with key regulatory roles in the associated processes or pathways .
thus, this suggests that the prmts studied here may regulate a common set of biological processes regulating directly or indirectly a few key major regulatory genes, which then control the expression of hundreds of genes associated with different biological responses .
our data resembles the results shown for muscle differentiation in mouse and zebrafish, where prmt <dig> and prmt <dig> positively regulate the expression of genes involved in myogenesis  <cit> .
therefore, the results of our genome-wide analysis do not support the idea that prmt <dig> and prmt <dig> act specifically as transcriptional repressor and activator, respectively, as previously suggested based on the analysis of a few genes.
in fact, there is an increasing number of publications revealing roles for prmt <dig> as a transcriptional activator and roles for prmt <dig> as a transcriptional repressor  <cit> .
it is worth mentioning that we cannot determine if the changes in mrna levels observed between wild-type and prmt mutant plants are mediated by alterations in histone methylation, or by changes in the methylation status of non-histone targets that regulate gene expression at the transcriptional and/or post-transcriptional levels.
impact of prmt <dig> and prmt4s on genome-wide as
several reports have previously suggested a key role for type i and type ii arginine methyltransferases in the regulation of pre-mrna splicing.
indeed, the role of prmt <dig> in this process has been well supported by genome-wide analyses of pre-mrna splicing in prmt <dig> mutant plants as well as in mammalian cells with reduced prmt <dig> expression.
in contrast, our knowledge of the role of prmt <dig> on pre-mrna splicing is still limited to its effect on a few individual splicing events  <cit> .
to characterize and compare the roles of arabidopsis prmt <dig> and prmt <dig> on pre-mrna splicing, we evaluated their effects on annotated as events from genes expressed above a minimal threshold level in all genotypes.
we found significant alterations in  <dig> as events in prmt <dig> mutants and in  <dig> in prmt4a;4b mutants, representing  <dig> and 21% of all as events evaluated, respectively.
among the altered as events identified,  <dig> exhibited increased inclusion and  <dig> decreased inclusion simultaneously in both mutants .
no significant differences were observed in the distribution of the as categories 5′ and 3′ alternative splicing site, intron retention and exon skipping .
the as events affected in common in both mutants were classified into functional categories, as described for the expression analysis, and evaluated for enrichment of specific categories in this data-set relative to their frequency in the genome.
we found a significant over-representation for categories such as primary metabolism, response to light, response to hormones and abiotic stress .
in addition, we found alterations in specific genes associated with salt stress, rna processing/splicing and flowering time regulation , which could be at least partially responsible for the mutant phenotypes .
it is worth mentioning that some of the as changes observed were specific for each mutant.
in particular, the alteration in as previously reported for the clock gene prr <dig>   <cit>  was clearly observed in prmt <dig> but not in prmt4a;b mutants .
indeed, a change in as of prr <dig> is thought to be responsible for the circadian defect present in prmt <dig> mutants  <cit> .
lack of effect of prmt4s on this as event is therefore consistent with the absence of a circadian phenotype in this mutant.
taken together, both mutants analyzed displayed significant alterations in the regulation of annotated as events highlighting a key role for both prmt <dig> and prmt <dig> in the regulation of as.
in contrast to what we observed for gene expression, we did not find an over-representation of genes related to rna processing among those affected at the as level in the prmt <dig> and prmt4a;4b mutants, suggesting that alterations in this process most likely result from effects on the expression of genes encoding splicing factors as well as from the post-translational regulation of specific proteins involved in rna processing.
on the other hand, processes such as primary metabolism, translation, response to light, hormone responses, and abiotic stress tolerance, all seem to be affected through changes in both gene expression and as.figure  <dig>
impact of prmt <dig> and prmt4s on genome wide as.
overlap between significantly altered alternative splicing events.
representation factor of alternative splicing events co- regulated by prmt <dig> and prmt4s in the selected functional categories.
*: indicates over-representation compared to random.
representation factor significance was assessed using a hypergeometric test .
alternative splicing events altered in
prmt5
and
prmt4a;4b
.
splicing ratio of 3′ alternative splicing events affected in both mutants.
splicing ratio of 5′ alternative splicing events altered in both mutants.
splicing ratio of intron retention events affected in both mutants.
splicing ratio of exon skipping events altered in both mutants.
the go category to which each gene belongs is indicated at the bottom of each panel.
analysis of the effects of prmt <dig> and prmt4s on constitutive pre-mrna splicing
to evaluate the role of prmt <dig> and prmt <dig> on constitutive splicing, we characterized the impact of mutations in these genes on splicing of all introns not annotated as alternatively spliced, which are present in genes expressed above a threshold level in all genotypes .
we found  <dig> introns with increased retention in prmt <dig> and  <dig> in prmt4a;4b mutants relative to wild-type plants.
this represents  <dig>  and  <dig> % of all introns studied in prmt <dig> and prmt4a;4b mutants, respectively.
interestingly, a much larger effect was observed in introns annotated as alternatively spliced, with  <dig> % of these affected in prmt <dig> mutants and  <dig> % in prmt4a;4b mutants.
thus, these results clearly indicate that prmt <dig> and prmt4s have much larger impact on alternative compared to constitutive splicing, as was previously reported for prmt <dig> in arabidopsis based on data from tiling arrays and a hr rt-pcr panel of well characterized as events, in contrast to what was observed for mammals  <cit> .
interestingly, many of the increased intron retention events identified were similarly affected in prmt <dig> and prmt4a;4b mutants , as shown for gene expression and as.
we then categorized these common set of intron retention events using gene ontology, and found a significant enrichment in the categories corresponding to primary metabolism, response to hormones and biotic stress .
we also found several intron retention events associated with genes involved in the regulation of rna splicing, light signaling, flowering, hormone signaling, and abiotic/biotic stress .
this data set supports the idea that prmt <dig> and prmt <dig> regulate the aforementioned processes, at least in part, through their effects on the regulation of pre-mrna splicing of genes associated with them.figure  <dig>
analysis of the effects of prmt <dig> and prmt4s on constitutive pre-mrna splicing.
overlap between introns with increased retention in both mutants.
representation factor of introns whose retention increased simultaneously in prmt <dig> and prmt4a;4b in the selected functional categories.
*: indicates over-representation compared to random, representation factor significance was assessed using a hypergeometric test .
splicing ratios of some representative events showing increased inclusion in both mutants is displayed .
the go category to which each gene belongs is indicated at the bottom of each panel.
then, we validated alterations in pre-mrna splicing detected using rna-seq for three splicing events.
one of this was an intron retention event associated with the gene at3g <dig>  which was similarly affected in both prmt <dig> as well as prmt4a;4b mutants .
another event validated was an intron retention event associated with the core clock gene at2g <dig>  also known as prr <dig>  which increased in prmt <dig> and was not affected in prmt4a;4b .
finally, we assessed a multiple splicing event that was affected only in prmt4a;4b at the gene at5g <dig>  which exhibited an increased retention of the intron  <dig> reported as constitutively spliced and, simultaneously, displayed an increased retention of the exon  <dig> that is reported as an alternative exon skipping event .figure  <dig>
rt-pcr analysis of alternative splicing events.
rt-pcr validation of three events identified through rna-seq.
event affected in both mutants simultaneously.
event affected only in prmt <dig>   event affected only in prmt4a;4b.
the read density map for each event evaluated is displayed .
a scheme describing each gene is displayed below the read density maps, with exons and introns displayed as boxes and lines respectively.
a red square encloses the measured event, while green arrows display the position of the oligo used for the rt-pcr measurement.
an image of the agarose gel with the rt- pcr amplicons is displayed next to the read density maps.
black arrows indicate amplicon sizes.
+: retrotranscriptase added, −: retrotranscriptase not added, gdna: genomic dna control.
each image represents one of three biological replicates measured.
finally, we evaluated whether there was any change in the splice-site sequences of the intron retention events affected in prmt mutants compared to the consensus splice-site sequence of all introns present in the arabidopsis genome .
interestingly, as previously reported for prmt <dig> mutants  <cit> , we found that the donor splice site sequences of the splicing events affected only in prmt <dig> mutants, or simultaneously altered in prmt <dig> and prmt4a;b mutants, displayed an under-representation of the consensus a and g nucleotides present in the − <dig> and − <dig> positions of the consensus donor splice site .
this indicates that the splicing events predominantly regulated by prmt <dig> alone, or simultaneously by prmt <dig> or prmt <dig>  are enriched in weak splice sites that deviate from the consensus sequence.
therefore, prmt <dig> and prmt <dig> are likely to regulate pre-mrna splicing, at least in part, by contributing to stabilizing weak rna-rna interactions between donor splice sites that deviate from the consensus sequence, and the sequence present in the u <dig> snrna that is complementary to the consensus donor splice-site sequence.
on the other hand, no deviation from the consensus sequence was observed for the acceptor splice site , or for the donor splice site of the events affected only in prmt <dig> mutants .
this observation suggests that prmt <dig> may also contribute to the regulation of a subset of pre-mrna splicing events acting, at least in part, by a different mechanism than prmt <dig> figure  <dig>
bioinformatic analysis of donor splice-site sequences.
pictograms showing the frequency distribution of nucleotides at the 5′ splice site of   <dig>  gt_ag_u <dig> arabidopsis introns,  the most significantly intron retention events whose splicing were altered simultaneously in both prmt <dig> and prmt4a;4b,  the most significantly intron retention events altered only in prmt <dig> and  the most significantly intron retention events altered only in prmt4a;4b.
the representation factor  is the frequency in the data set of interest divided by the total frequency.
for each rf a p-value was calculated using the hypergeometric test.
CONCLUSIONS
our comparative analysis of the physiological and molecular alterations present in arabidopsis prmt <dig> and prmt4a;4b mutants clearly shows that, in plants, the type ii arginine methyltransferase prmt <dig> and the type i methyltransferases prmt4a and 4b most often co-regulate the same biological processes, affecting them in a similar manner.
at the same time, we also found that some physiological processes are specifically affected by prmt <dig> but not by prmt4s.
the transcriptome analysis conducted with rna-seq revealed that the co-regulated nature of most of the physiological processes evaluated was more likely the result of overlapping roles of prmt <dig> and prmt <dig> in the regulation of transcription and pre-mrna splicing.
thus, our findings suggest that the general idea that prmt <dig> and prmt <dig> play antagonist roles in the regulation of gene expression, may probably be a biased conclusion based on the analysis of few genes.
our study, besides being the first genome wide analysis of the effects of prmt <dig> on gene expression and pre-mrna splicing, strongly suggest that prmt <dig> and prmt <dig> have mostly similar molecular functions regulating gene expression at the transcriptional and post-transcriptional level.

 post-translational modification of proteins is a typical mark of signal transduction pathways through which organisms are able to react quickly to changes in their surrounding by expanding the structural and functional diversity of the proteome. arginine methylation is a common post- translational modification in eukaryotic organisms, and is catalyzed by a family of enzymes known as protein arginine methyltransferases. this post-translational modification modulates a myriad of cellular processes through its effects on proteins involved in the regulation of chromatin structure, transcription, rna processing, signal transduction and cellular differentiation, among other processes. prmts are classified in four groups: type i prmts that generate ω-ng-monomethyl arginine and ω-ng,ng-asymmetric dimethylarginines, type ii prmts that generate ω-ng-monomethyl arginine and ω-ng,ng-symmetric dimethylarginines, type iii prmts that generate only ω-ng-monomethyl arginine, and type iv that generate only δ-ng-monomethyl arginine. currently, four different genes encoding prmts have been described in saccharomyces cerevisiae, eight in oryza sativa, and nine in humans, drosophila and arabidopsis thaliana. in mammals, there are six well characterized arginine methyltransferases, five of them corresponding to type i prmts; while the only type ii prmt known so far is prmt it has been proposed that there is no major redundancy between these enzymes, at least in mammals, since knock-out mice for each prmt display clearly different phenotypes. it is known that proteins that possess glycine and arginine- rich motifs are often targets of prmts. transcriptional regulation by methylation of histones and non-histone proteins is one of the more characterized functions of prmts. it has been reported that prmt methylates arg of histone h prmt methylates arg arg and arg of histone h and that prmt methylates arg of histone h and arg of histone h in mammals, formation of adma in histones by prmt and prmt participates in gene activation while formation of sdma by prmt is associated with gene repression. arginine methylation has also been associated with the regulation of the initiation and elongation steps of transcription. the recruitment of prmt to transcriptional promoters results in methylation of histone acetyltransferases resulting in a positive effect on transcription, while prmt methylates the transcriptional elongation factor spt regulating its interaction with rna polymerase ii. another well characterized role of prmts is the regulation of rna processing. rna binding proteins fulfill numerous tasks ensuring the proper processing and folding as well as the stabilization and localization of rnas and mrna translation. these proteins represent major targets for prmts because most hnrnps possess gar motifs. prmts are also known to methylate sm spliceosomal proteins b, b’, d and d and this mediates their assembly into mature small nuclear ribonucleoprotein particles, and has been associated with a role for prmt in the regulation of pre-mrna splicing. on the other hand, prmt regulates the coupling of transcription and mrna processing through methylation of splicing factors. strikingly, only a few studies have compared side by side the roles of distinct prmts in the regulation of different physiological and molecular processes. in fact, several of these studies showed that prmt and prmt simultaneously control myogenesis, with both proteins having similar positive roles in the control of expression of genes known to play a key role regulating this developmental process. this is at odds with the current view that assumes that prmt a type i prmt, acts as a co-activator of gene expression, while prmt the main type ii prmt, acts as a transcriptional repressor. whether the similar positive role that prmt and prmt play in the control of myogenesis is the exception or the rule is not known. discriminating between these two alternatives requires a side-by-side comparison of the effects of prmt and prmt on gene expression at a genome-wide level. interestingly, both prmt and prmt have been proposed to play key roles in the regulation of pre-mrna splicing. as previously reviewed, this regulatory role may be exercised through direct methylation of core spliceosomal proteins or, alternatively or in addition, through regulation of the coupling between transcription and mrna processing. however, while genome-wide analyses of pre-mrna splicing have been conducted to characterize prmt deficient plants or animals, similar global analysis are missing for mutants affected in prmt plants are ideal organisms to conduct a side-by-side comparison of the roles of prmt and prmt on gene expression and rna processing, because mutant plants lacking these proteins are viable and fertile, while defects in these two genes are lethal in mammals. in arabidopsis thaliana there are seven type i arginine methyltransferases and, as in mammals, one type ii enzyme. to date, the most intensively studied prmt in plants is prmt which controls flowering time, circadian rhythms photomorphogenic development and salt tolerance acting on epigenetic regulation of gene expression and on pre-mrna splicing of a sub-set of genes. regarding type i prmts, it has been shown that prmt prmt1b, and prmt4a together with prmt4b, are all involved in the regulation of flowering time. both type i and type ii prmts appear to regulate flowering time in arabidopsis thaliana through effects on flc expression. flc is a mads box transcription factor that represses flowering, and prmt prmt mutants and the prmt4a;4b double mutant all exhibit increased flc expression, which is partially responsible for the delayed flowering observed in these mutant plants. so far, the only phenotype that has been reported for prmt4a;4b mutant plants is delayed flowering. interestingly, this mutant resembles prmt plants at the morphological level, showing some degree of growth retardation and dark green leaves. however, whether prmt4a;4b also exhibits other physiological alterations present in prmt mutants such as defects in circadian clock function, photomorphogenic development or salt stress tolerance is not known. in this study we compared side by side the role of prmt and prmt4s in the regulation of several physiological processes in arabidopsis thaliana, and coupled this with a genome-wide comparison of their effects on gene expression and pre-mrna splicing using rna-seq. we found that prmt and prmt4a;4b mutants not only displayed similar alterations in flowering time regulation, but also exhibited reduced inhibition of hypocotyl elongation under both red and blue light, revealing defects in light signaling. in addition, similarly to what has been reported for prmt mutants, we found that prmt4a;4b double mutants exhibited reduced tolerance to salt stress. however, in contrast to what is observed in prmt mutants, the prmt4a;prmt4b double mutant did not exhibit alterations in circadian rhythms. rna-seq data showed that both mutants display similar alterations in the expression of genes related to: transcription, mrna processing, mrna splicing, translation, light signaling, response to hormones and both abiotic and biotic stress. in addition, we also found that these mutants have similar alterations in alternative splicing of genes related to translation, light signaling, response to hormones and both abiotic and biotic stress. finally, we observed a significant number of novel intron retention events in both mutants, revealing alterations in a subset of constitutive as well as as events in those plants. hence, this study shows that in arabidopsis thaliana, type i prmt4s and type ii prmt regulate overlapping as well as distinct physiological processes, most likely through similar effects on gene expression and pre-mrna splicing of a subset of key regulatory genes. additionally, this study is the first to report an analysis of the role of prmt4s on pre-mrna splicing at a global level.  prmt and prmt control overlapping as well as distinct physiological processes before conducting a genome-wide comparison of the roles of prmt and prmt in the regulation of gene expression and rna processing in arabidopsis thaliana, we performed a side by side analysis of the effect of these genes on the control of several developmental and physiological processes. in particular, we focused the analysis on clock-associated processes, such as flowering time regulation, photomorphogenic responses and circadian rhythms, as well as on salt stress tolerance. all these physiological and developmental processes have previously been shown to be affected in prmt mutants but have not been characterized in prmt4a;4b mutants, with the exception of flowering time. indeed, as previously reported individually for prmt and prmt4a;4b,, both mutants showed a late flowering phenotype compared to wild-type plants of the col- accession. however, while both mutants displayed a clear and similar late flowering phenotype under short day photoperiods, the late flowering phenotype of prmt was much stronger than that of prmt4a;4b mutant plants under long day photoperiods.figure side by side comparison of the physiological roles of prmt and prmt flowering time measured as the number of rosette leaves at bolting in long day and short day photoperiods. anova followed by a tukey’s multiple comparision test was used to evaluate the statistical significance of the differences observed between genotypes. errors bars indicate sd. circadian rhythms of leaf movement in continuous light. period of the circadian rhythms was estimated with brass software. hypocotyl length of seedlings grown under continuous red light or continuous blue light. comparison of root growth on ms medium with 100 mm nacl. root growth was measured relative to controls. more than roots were measured for each data point. data represents the mean with sd of three independent experiments. salt tolerance was assessed through the analysis of survival rate on ms medium containing 160 mm nacl. data represents the mean with sd of three independent experiments. we and others have previously shown that prmt plays a key role in the regulation of circadian rhythms in arabidopsis, but whether prmt contributes to the regulation of clock function is not known. to evaluate this we monitored circadian rhythms in leaf movements in wild-type plants, prmt and prmt4a;4b mutant plants. interestingly, while prmt mutant plants showed the previously described long period phenotype for leaf movement, the rhythms observed in prmt4a;4b mutants were similar to those of wild-type plants. in order to analyze the role of these prmts in photomorphogenesis we evaluated light inhibition of hypocotyl elongation during de-etiolation in seedlings exposed to different fluence rates of red and blue light. we found that both prmt and prmt4a;4b mutants were hyposensitive to red light at all fluence rates tested. a similar phenotype was observed for both mutants under blue light as well. prmt has been shown to regulate salt stress tolerance in arabidopsis. to test if prmt4a, together with prmt4b, were also involved in the regulation of this physiological response, we analyzed root growth, a process affected by high salt concentrations. root length of wild-type plants grown on ms medium containing 100 mm nacl was approximately 80% of that shown by wild-type plants grown on ms medium alone. in contrast, root length of prmt and prmt4a;4b mutants grown on medium containing the same concentration of salt was only 30% and 50%, respectively, relative to that of plants from these genotypes grown on ms medium. we also conducted a survival rate assay and found that growth of both prmt and prmt4a;4b mutants was almost completely inhibited in medium containing 160 mm nacl, while wild-type plants displayed only a slightly inhibited growth rate under this condition. altogether this shows that prmt and, to a lesser extent prmt4a, together with prmt4b, are all involved in the control of salt stress tolerance in arabidopsis. impact of prmt and prmt4s on genome wide gene expression in order to study the extent of the regulatory impact of prmt and prmt4s on gene expression we analyzed the transcriptome of wild-type, prmt and prmt4a;b plants grown under standard non-stressful conditions using rna-seq. we found genes over-expressed and under-expressed in prmt mutants, as well as genes over-expressed and under-expressed in prmt4a;4b mutants, relative to wild-type plants. strikingly, many of the differentially expressed genes were similarly affected in both mutants, with and genes over-expressed or under-expressed in common, respectively. on the other hand, only genes, out of a total of and genes differentially expressed in either prmt or prmt4a;b, respectively, were antagonistically affected in the mutants. therefore, it is highly unlikely that the prmts analyzed here exhibit opposite biological and molecular roles.figure impact of prmt and prmt on genome wide gene expression. overlap between differentially expressed genes in both prmt and prmt4a;4b mutants. representation factor of genes co- regulated by prmt and prmt4s in the selected functional categories. *: indicates over-representation compared to random. significance was assessed using a hypergeometric test. log fold change of representative genes co- regulated by prmt and prmt4s. the differentially expressed genes that were similarly affected in both mutants were categorized into functional groups based on gene ontology. fifteen functional categories of our interest were examined in detail determining the representation factor for each category. the representation factor is the number of overlapping genes observed divided by the expected number of overlapping genes drawn randomly from two independent groups. among the up-regulated genes, we found a significant enrichment for categories corresponding to transcription, mrna processing, rna splicing, response to light, response to hormones, abiotic stress and biotic stress. for down-regulated genes, we found significant enrichment for genes associated with primary metabolism, translation, response to light, abiotic stress and biotic stress. none of the categories studied here displayed a statistically significant under-representation. finally, we also found a number of genes that were significantly affected only in prmt or prmt4a;b mutants, some of which may be responsible for the partially distinct phenotypes of the mutants, such as the differential effect on circadian rhythmicity. altogether, our physiological and molecular analysis suggests that these prmts regulate most of the mentioned processes controlling the expression of a common set of genes. indeed, when we analyzed in detail the identity of the co-regulated genes associated with the biological processes mentioned above, we found that many of them corresponded to transcription factors with key regulatory roles in the associated processes or pathways. thus, this suggests that the prmts studied here may regulate a common set of biological processes regulating directly or indirectly a few key major regulatory genes, which then control the expression of hundreds of genes associated with different biological responses. our data resembles the results shown for muscle differentiation in mouse and zebrafish, where prmt and prmt positively regulate the expression of genes involved in myogenesis. therefore, the results of our genome-wide analysis do not support the idea that prmt and prmt act specifically as transcriptional repressor and activator, respectively, as previously suggested based on the analysis of a few genes. in fact, there is an increasing number of publications revealing roles for prmt as a transcriptional activator and roles for prmt as a transcriptional repressor. it is worth mentioning that we cannot determine if the changes in mrna levels observed between wild-type and prmt mutant plants are mediated by alterations in histone methylation, or by changes in the methylation status of non-histone targets that regulate gene expression at the transcriptional and/or post-transcriptional levels. impact of prmt and prmt4s on genome-wide as several reports have previously suggested a key role for type i and type ii arginine methyltransferases in the regulation of pre-mrna splicing. indeed, the role of prmt in this process has been well supported by genome-wide analyses of pre-mrna splicing in prmt mutant plants as well as in mammalian cells with reduced prmt expression. in contrast, our knowledge of the role of prmt on pre-mrna splicing is still limited to its effect on a few individual splicing events. to characterize and compare the roles of arabidopsis prmt and prmt on pre-mrna splicing, we evaluated their effects on annotated as events from genes expressed above a minimal threshold level in all genotypes. we found significant alterations in as events in prmt mutants and in in prmt4a;4b mutants, representing and 21% of all as events evaluated, respectively. among the altered as events identified, exhibited increased inclusion and decreased inclusion simultaneously in both mutants. no significant differences were observed in the distribution of the as categories 5′ and 3′ alternative splicing site, intron retention and exon skipping. the as events affected in common in both mutants were classified into functional categories, as described for the expression analysis, and evaluated for enrichment of specific categories in this data-set relative to their frequency in the genome. we found a significant over-representation for categories such as primary metabolism, response to light, response to hormones and abiotic stress. in addition, we found alterations in specific genes associated with salt stress, rna processing/splicing and flowering time regulation, which could be at least partially responsible for the mutant phenotypes. it is worth mentioning that some of the as changes observed were specific for each mutant. in particular, the alteration in as previously reported for the clock gene prr was clearly observed in prmt but not in prmt4a;b mutants. indeed, a change in as of prr is thought to be responsible for the circadian defect present in prmt mutants. lack of effect of prmt4s on this as event is therefore consistent with the absence of a circadian phenotype in this mutant. taken together, both mutants analyzed displayed significant alterations in the regulation of annotated as events highlighting a key role for both prmt and prmt in the regulation of as. in contrast to what we observed for gene expression, we did not find an over-representation of genes related to rna processing among those affected at the as level in the prmt and prmt4a;4b mutants, suggesting that alterations in this process most likely result from effects on the expression of genes encoding splicing factors as well as from the post-translational regulation of specific proteins involved in rna processing. on the other hand, processes such as primary metabolism, translation, response to light, hormone responses, and abiotic stress tolerance, all seem to be affected through changes in both gene expression and as.figure impact of prmt and prmt4s on genome wide as. overlap between significantly altered alternative splicing events. representation factor of alternative splicing events co- regulated by prmt and prmt4s in the selected functional categories. *: indicates over-representation compared to random. representation factor significance was assessed using a hypergeometric test. alternative splicing events altered in prmt5 and prmt4a;4b. splicing ratio of 3′ alternative splicing events affected in both mutants. splicing ratio of 5′ alternative splicing events altered in both mutants. splicing ratio of intron retention events affected in both mutants. splicing ratio of exon skipping events altered in both mutants. the go category to which each gene belongs is indicated at the bottom of each panel. analysis of the effects of prmt and prmt4s on constitutive pre-mrna splicing to evaluate the role of prmt and prmt on constitutive splicing, we characterized the impact of mutations in these genes on splicing of all introns not annotated as alternatively spliced, which are present in genes expressed above a threshold level in all genotypes. we found introns with increased retention in prmt and in prmt4a;4b mutants relative to wild-type plants. this represents and % of all introns studied in prmt and prmt4a;4b mutants, respectively. interestingly, a much larger effect was observed in introns annotated as alternatively spliced, with % of these affected in prmt mutants and % in prmt4a;4b mutants. thus, these results clearly indicate that prmt and prmt4s have much larger impact on alternative compared to constitutive splicing, as was previously reported for prmt in arabidopsis based on data from tiling arrays and a hr rt-pcr panel of well characterized as events, in contrast to what was observed for mammals. interestingly, many of the increased intron retention events identified were similarly affected in prmt and prmt4a;4b mutants, as shown for gene expression and as. we then categorized these common set of intron retention events using gene ontology, and found a significant enrichment in the categories corresponding to primary metabolism, response to hormones and biotic stress. we also found several intron retention events associated with genes involved in the regulation of rna splicing, light signaling, flowering, hormone signaling, and abiotic/biotic stress. this data set supports the idea that prmt and prmt regulate the aforementioned processes, at least in part, through their effects on the regulation of pre-mrna splicing of genes associated with them.figure analysis of the effects of prmt and prmt4s on constitutive pre-mrna splicing. overlap between introns with increased retention in both mutants. representation factor of introns whose retention increased simultaneously in prmt and prmt4a;4b in the selected functional categories. *: indicates over-representation compared to random, representation factor significance was assessed using a hypergeometric test. splicing ratios of some representative events showing increased inclusion in both mutants is displayed. the go category to which each gene belongs is indicated at the bottom of each panel. then, we validated alterations in pre-mrna splicing detected using rna-seq for three splicing events. one of this was an intron retention event associated with the gene at3g which was similarly affected in both prmt as well as prmt4a;4b mutants. another event validated was an intron retention event associated with the core clock gene at2g also known as prr which increased in prmt and was not affected in prmt4a;4b. finally, we assessed a multiple splicing event that was affected only in prmt4a;4b at the gene at5g which exhibited an increased retention of the intron reported as constitutively spliced and, simultaneously, displayed an increased retention of the exon that is reported as an alternative exon skipping event.figure rt-pcr analysis of alternative splicing events. rt-pcr validation of three events identified through rna-seq. event affected in both mutants simultaneously. event affected only in prmt event affected only in prmt4a;4b. the read density map for each event evaluated is displayed. a scheme describing each gene is displayed below the read density maps, with exons and introns displayed as boxes and lines respectively. a red square encloses the measured event, while green arrows display the position of the oligo used for the rt-pcr measurement. an image of the agarose gel with the rt- pcr amplicons is displayed next to the read density maps. black arrows indicate amplicon sizes. +: retrotranscriptase added, −: retrotranscriptase not added, gdna: genomic dna control. each image represents one of three biological replicates measured. finally, we evaluated whether there was any change in the splice-site sequences of the intron retention events affected in prmt mutants compared to the consensus splice-site sequence of all introns present in the arabidopsis genome. interestingly, as previously reported for prmt mutants, we found that the donor splice site sequences of the splicing events affected only in prmt mutants, or simultaneously altered in prmt and prmt4a;b mutants, displayed an under-representation of the consensus a and g nucleotides present in the − and − positions of the consensus donor splice site. this indicates that the splicing events predominantly regulated by prmt alone, or simultaneously by prmt or prmt are enriched in weak splice sites that deviate from the consensus sequence. therefore, prmt and prmt are likely to regulate pre-mrna splicing, at least in part, by contributing to stabilizing weak rna-rna interactions between donor splice sites that deviate from the consensus sequence, and the sequence present in the u snrna that is complementary to the consensus donor splice-site sequence. on the other hand, no deviation from the consensus sequence was observed for the acceptor splice site, or for the donor splice site of the events affected only in prmt mutants. this observation suggests that prmt may also contribute to the regulation of a subset of pre-mrna splicing events acting, at least in part, by a different mechanism than prmt figure bioinformatic analysis of donor splice-site sequences. pictograms showing the frequency distribution of nucleotides at the 5′ splice site of gt_ag_u arabidopsis introns, the most significantly intron retention events whose splicing were altered simultaneously in both prmt and prmt4a;4b, the most significantly intron retention events altered only in prmt and the most significantly intron retention events altered only in prmt4a;4b. the representation factor is the frequency in the data set of interest divided by the total frequency. for each rf a p-value was calculated using the hypergeometric test.  our comparative analysis of the physiological and molecular alterations present in arabidopsis prmt and prmt4a;4b mutants clearly shows that, in plants, the type ii arginine methyltransferase prmt and the type i methyltransferases prmt4a and 4b most often co-regulate the same biological processes, affecting them in a similar manner. at the same time, we also found that some physiological processes are specifically affected by prmt but not by prmt4s. the transcriptome analysis conducted with rna-seq revealed that the co-regulated nature of most of the physiological processes evaluated was more likely the result of overlapping roles of prmt and prmt in the regulation of transcription and pre-mrna splicing. thus, our findings suggest that the general idea that prmt and prmt play antagonist roles in the regulation of gene expression, may probably be a biased conclusion based on the analysis of few genes. our study, besides being the first genome wide analysis of the effects of prmt on gene expression and pre-mrna splicing, strongly suggest that prmt and prmt have mostly similar molecular functions regulating gene expression at the transcriptional and post-transcriptional level. 
our global analysis indicates that two of the most important type i and type ii arginine methyltransferases, prtm and prmt have mostly overlapping as well as specific, but not opposite, roles in the global regulation of gene expression in plants. we found that prmt and prmt4a;4b mutants showed similar alterations in flowering time, photomorphogenic responses and salt stress tolerance, while only prmt mutants exhibited alterations in circadian rhythms. arginine methylation is catalyzed by a family of enzymes known as protein arginine methyltransferases. methylation at arginine residues is an important post-translational modification that regulates a myriad of essential cellular processes in eukaryotes, such as transcriptional regulation, rna processing, signal transduction and dna repair. furthermore, prmt and prmt4s co-regulated the expression and splicing of key regulatory genes associated with transcription, rna processing, responses to light, flowering, and abiotic stress tolerance, being candidates to mediate the physiological alterations observed in the mutants. prmts are classified as type i or type ii, depending on the position of the methyl group on the guanidine of the methylated arginine. here we compared side by side the physiological and molecular roles of the best characterized plant prmts, the type ii prmt and the type i prmt also known as carm in mammals.
 methylation at arginine residues is an important post-translational modification that regulates a myriad of essential cellular processes in eukaryotes, such as transcriptional regulation, rna processing, signal transduction and dna repair. arginine methylation is catalyzed by a family of enzymes known as protein arginine methyltransferases. prmts are classified as type i or type ii, depending on the position of the methyl group on the guanidine of the methylated arginine. previous reports have linked symmetric r methylation to transcriptional repression, while asymmetric r methylation is generally associated with transcriptional activation. however, global studies supporting this conclusion are not available.  here we compared side by side the physiological and molecular roles of the best characterized plant prmts, the type ii prmt and the type i prmt also known as carm in mammals. we found that prmt and prmt4a;4b mutants showed similar alterations in flowering time, photomorphogenic responses and salt stress tolerance, while only prmt mutants exhibited alterations in circadian rhythms. an rna-seq analysis revealed that expression and splicing of many differentially regulated genes was similarly enhanced or repressed by prmt and prmt4s. furthermore, prmt and prmt4s co-regulated the expression and splicing of key regulatory genes associated with transcription, rna processing, responses to light, flowering, and abiotic stress tolerance, being candidates to mediate the physiological alterations observed in the mutants.  our global analysis indicates that two of the most important type i and type ii arginine methyltransferases, prtm and prmt have mostly overlapping as well as specific, but not opposite, roles in the global regulation of gene expression in plants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 