BACKGROUND
the current view of cellular rna organization indicates an rna infrastructure  <cit> , which describes the spatial and temporal network of the many different rnas.
the interconnection of the rna-processing pathways is crucial for cellular processes because different rna processing mechanisms are tightly linked.
for example, transcription and splicing happen in close proximity  <cit> , and splicing is tightly connected with downstream mrna processes including localization, translational regulation, and nonsense-mediated decay  <cit> .
with the advancement of large-scale rna analysis and high-throughput sequencing, conserved features of the eukaryotic rna infrastructure have come to light from studies in animals and plants.
in contrast to the genome-wide transcriptional information known in other eukaryotic models, only limited information on rna biology is available from g. intestinalis and t. vaginalis.
we can compare the ncrnas from these two protists with other model eukaryotes , to understand more about the general nature of the rna infrastructure within eukaryotes.
previous studies in giardia have identified key eukaryotic ncrnas such as the rnase p  <cit> , snornas  <cit> , spliceosomal snrnas  <cit> , mirnas  <cit>  and antisense transcripts  <cit> .
studies on trichomonas ncrnas show that the currently known ncrnas also exhibit typical features of eukaryotes  <cit>  including rnase p  <cit> , rnase mrp  <cit> , snrnas  <cit>  and some snornas  <cit> .
however, there are still gaps in our knowledge of rna processing in these species, especially in the characterization of the rnase mrp rna in giardia, and the different types of snornas in trichomonas .
annotations of the rnase p rna are also not clear in these genomes.
in this study, we characterize medium length ncrnas including rnase mrp, rnase p and snornas from two protozoan parasites: giardia intestinalis  and trichomonas vaginalis , to clarify uncertainties about conserved features of the rna infrastructure.
we have previously sequenced ncrnas sized from  <dig> to  <dig> nucleotides  from g. intestinalis and t. vaginalis, and identified from this data small ncrnas such as micrornas in the two protists  <cit> .
using this same sequencing data we are also able to analyze longer ncrnas due to the wide size range.
in this study we identify medium-length ncrnas  from small rna based sequencing data, characterize the rnase mrp of g. intestinalis, and clarify annotations of rnase p and mrp from both g. intestinalis and t. vaginalis.
we also identify new h/aca box snornas from these species.
our study clearly demonstrates that high-throughput sequencing cannot only screen for small regulatory rnas, but can also be used for the characterization of longer ncrnas from diverse organisms.
results from our work support that g. intestinalis and t. vaginalis possess a rich network of rna processing components expected in the consensus eukaryotic rna infrastructure.
RESULTS
construction of rna contigs using consensus mapping
the total rnas from g. intestinalis and t. vaginalis were purified, size fractionated and sequenced according to methods.
constructing contigs of short to medium rnas requires a different approach from standard assembly protocols.
most ncrnas do not have long poly-a tails; therefore small-rna sequencing is a way of recovering medium-length ncrnas when used in combination with consensus mapping.
we successfully generated rna contigs from g. intestinalis and t. vaginalis and compared the updated mapping software bowtie  <cit>  to the original software maq  <cit> .
overall, the contigs constructed using bowtie were longer compared to the ones constructed using maq, although the overall number of contigs was fewer .
*mpileup is part of the samtools package
with the length cutoff of the new contig datasets set above 50nt we discarded mature mirnas and sirnas.
de novo assembly tools such as velvet  <cit>  and abyss  <cit>  produced very few contigs and therefore were not used in this study.
hence, we recommend that for medium length ncrna assembly that a reference genome is used for the initial assembly until tools are developed to permit the de novo assembly of small contigs.
our study was carried out on g. intestinalis wb strain , and the genome assemblages of the other two strains  were used for comparison in some of the subsequent analysis.
our rna contigs and trimmed sequences were compared to the available g. intestinalis and t. vaginalis genome annotation to check that rrnas and trnas were represented as expected.
from the g. intestinalis sequences all  <dig> annotated rrnas and trnas from the wb isolate were covered by contigs generated with bowtie mapping.
however, many of these contigs contain illumina short reads mapped to multiple sites in the genome and therefore were assigned equally to each of the possibilities.
contigs were trimmed in length from the 5' end, to a minimum length of  <dig> nt,  <dig> nt and  <dig> nt to use as sequence tags and mapped against the annotated ncrnas.
our original trimmed sequence datasets were also mapped against the annotated ncrnas.
all g. intestinalis and t. vaginalis rrnas were found by sequence and contigs.
in g. intestinalis all trnas and rrnas were found from assemblage a with  <dig> trnas not found by contig but found by sequence in assemblage b,   and four not found by sequence or contig .
a comparison against assemblage e  had  <dig> trnas  not found by sequence or contig.
given that our sequences came from the same strain as assemblage a, not finding some sequences in the other strains is not surprising.
t. vaginalis had many annotations for its rrnas and trnas and the majority were found by both contig and sequence .
results from comparing our contigs and sequences with known trnas and rrnas indicate that our method is assembling ncrnas effectively, and that such contigs can be used as sequence tags if trimmed to  <dig> or  <dig> nucleotides.
identification of the mrp rna from g. intestinalis
rnase mrp is a ribonucleoprotein complex, consisting of one ncrna and  ~ <dig> proteins.
it catalyzes the nucleotide cleavage reaction at the a <dig> site  on the pre-rrna transcript.
mrp rna is evolutionarily related to the eukaryotic rnase p rna which processes the 3'- end of pre-trna transcripts  <cit> .
the rnase p and mrp complexes share a number of common protein subunits  <cit> , and the secondary structures of rnase p and mrp rnas share a common backbone.
the rnase p rna has been identified in all eukaryotes and prokaryotes studied to date and shown to be one of the few rnas that have retained catalytic features  <cit> .
the rnase mrp is thought to have evolved before the ancestor of modern eukaryotes  <cit> , but previously missing evidence of the mrp rna from g. intestinalis  <cit>  imposed a question on the origin of mrp and rrna processing.
however the conserved a <dig> site on rrna-gene sequence has been identified in g. intestinalis, strongly suggesting the presence of g. intestinalis mrp rna  <cit> .
our contigs contained a sequence that with infernal  <dig>   <cit>  mapped to the conserved cr- <dig> consensus region of the p <dig> pseudoknot structure of the mrp core structure .
both the pseudoknot and the actual sequences of cr- <dig> and cr- <dig> regions have previously been shown to be evolutionarily conserved throughout most eukaryotes  <cit> .
sequence alignment of the cr- <dig> and downstream predicted cr- <dig> regions of g. intestinalis mrp and a representative of other eukaryotic mrps showed a high degree of sequence similarity .
upon further analysis, contigs upstream and downstream to the conserved cr- <dig> region were found that did not have enough overlap to permit a longer contig to be predicted computationally.
this region is highly conserved between the three isolates of g. intestinalis  and as expected, our sequence had 100% identity to isolate a .
comparing the alignment with the predicted secondary structure  indicates that half of the nucleotide differences occur in single stranded regions and all but one change at the beginning of helix p <dig> are either compensated with a change at the corresponding position or changed to permit g-u wobble pairing.
thus, it is unlikely that any of these changes will have a major impact on the overall rnase mrp rnp secondary structure.
all predicted rnase mrp rna and rnase p rnas from g. intestinalis and t. vaginalis are given in table  <dig>
giardia b
isolate gs
acgj01002297
25811- <dig>
giardia b
isolate gs
acgj01002916
916- <dig>
all genomes downloaded from giardiadb  and trichdb   <cit>
the entire predicted secondary structure of g. intestinalis mrp including these conserved regions is shown in figure 3a.
a closer look at the structure shows that the cr- <dig> region also has the previously identified conserved pattern "anagnna"  <cit>  where the three most conserved "a"s are present.
not unexpectedly given the reduced nature of the g. intestinalis genome, the length and structure of the g. intestinalis mrp rna makes it one of the shortest among studied eukaryotes.
in t. vaginalis the mrp rna is longer and has a more extended p <dig> helix .
structurally, the g. intestinalis mrp rna is closer to that of the microsporidium species such as nosema locustae and encephalitozoon cuniculi, which are known to have the shortest mrps  <cit> .
another important characteristic of the mrp rna is the p <dig> helix, which is a structure common between rnase mrp and p. the p <dig> helix associates with the protein pop <dig>  <cit> , and for mrp it is essential for the cleavage of pre-rrnas at the a <dig> site on the pre-ribosomal rna transcript  <cit> .
previous computational studies have already identified both the rnase p and rnase mrp rnas from t. vaginalis  <cit> .
the p <dig> helix of g. intestinalis rnase p and rnase mrp are both short and lack the intra-helical loop, however they do share a  <dig> nt sequence at the terminal loop, consistent with previous studies showing the same relation of p and mrp in a number of eukaryotes  <cit> .
comparison of the p <dig> helices of t. vaginalis rnase p and mrp also show a consensus sequence located at the intra-helical loop and the terminal loop regions .
the structure of helix p <dig> between g. intestinalis mrp and p is also conserved, although no sequence similarity is observed.
although we are concentrating on the rnase p and rnase mrp ncrna molecules from g. intestinalis and t. vaginalis, it is worth a quick mention of the important proteins associated with their ribonucleoprotein macromolecules.
rnase p and rnase mrp macromolecules share many of their proteins  <cit> , which have been characterized to small extent in g. intestinalis  <cit> .
some proteins such as the scaffolding protein pop <dig> <cit>  can be hard to identify in protists, due to the large amount of evolutionary distance between them and species from which these proteins are known, but other proteins such as pop <dig> are much more conserved across eukaryotes  <cit> .
for a more detailed analysis of the changes in the secondary structure of the rna components we will require further domain and protein structural analysis to understand how the rna and protein components have evolved together.
however, even without this detailed protein-rna analysis, we can see that our results strongly support the conservation of the structure-function relationship between rnase mrp and p within g. intestinalis and t. vaginalis.
our study also sought to clarify the annotation of the rnase mrp rna especially in the assemblage b from version  <dig>  of the genome.
in this annotation within contig acgj <dig>  the mrp rna is overlapping with a snorna which is in turn overlapping an area annotated as rnase p. the rnase mrp rna from g. intestinalis we have identified does not match this region, but instead this region corresponds to its close relative the rnase p rna.
corrected contigs and co-ordinates for the assemblage b genes are given in table  <dig>
although we have characterized the g. intestinalis mrp rna computationally, further molecular biology experimentation will be required before it can be functionally verified.
until then, we classify our sequences in g. intestinalis as computationally predicted.
new h/aca box snornas
small nucleolar rnas are a group of ncrnas of variable length , which are involved in processing of several types of transcripts  <cit> .
most of the known snornas belong to two classes, which are determined by evolutionarily conserved sequence elements: the c/d box and h/aca box  <cit> .
snornas exist in large numbers in eukaryotes.
in humans, there have been more than  <dig> snornas identified  <cit> , and in general have been shown to have a diverse range of locations and expression patterns  <cit> .
in our previous studies, we have characterized novel c/d box snornas in both g. intestinalis and t. vaginalis  <cit> .
c/d box snornas direct 2'-o methylation, and are relatively easy to identify based on conserved sequence elements and complementary binding to the target rnas.
h/aca box snornas direct pseudouridylation, and often exhibit more variable features due to their shorter length of conserved elements and discontinuous complementary target binding regions.
as a first step in identification of new h/aca box snornas, the rrnas of g. intestinalis and t. vaginalis were aligned with human rrnas to locate conserved pseudouridylation sites, which were then incorporated into the search for the complementary target-binding regions in candidate h/aca box snornas.
after generating the negative controls, the rna contigs were searched using the established parameters from control runs .
table  <dig> shows the scoring rules and results from subsequent analysis.
in total,  <dig> g. intestinalis and  <dig> t. vaginalis new snornas were identified using this method.
the rna sequences and corresponding genomic positions are in additional file  <dig> and additional file  <dig>  one set of the new g. intestinalis snornas , and two sets of the new t. vaginalis snornas  are overlapping in the same genomic region on the plus and minus strand respectively.
the structures of the target binding regions of new snornas are shown in figure  <dig>  candidates had either one or two stems, but all contain only one target-binding site.
candidates with two stems can have the target-binding site on either stem.
further analysis showed that their second stems do not have targets on rrnas despite having the conserved stem-loop structure upstream of the aca box.
†the percentage of control sequences with total scores below the cutoff score
*number of predicted candidates with total scores above the cutoff score set from control analysis
the new g. intestinalis snornas all have different target pseudouridylation sites on rrnas, whereas two of the t. vaginalis new snornas  share the same target .
in addition, gi/aca.
<dig>  tv/aca.
<dig> and tv/aca.
<dig> target a conserved site on the large subunit rrna, the same as gi/aca.
<dig> and tv/aca.
<dig>  the target regions are highly conserved between g. intestinalis and t. vaginalis, but the sequences of the snornas targeting these sites do not show substantial sequence similarity.
a blast  <cit>  search of the newly identified g. intestinalis h/aca box snornas was performed against the genomes of the other two isolates of giardia strains  to look for homologous sequences.
three g. intestinalis new snornas have homologous sequences in either assemblage b or e, and  <dig> have homologous sequences in both assemblage b and e. overall the sequences are highly conserved and the nucleotides complementary to the rrna target sequences show minimal changes across the three giardia strains.
discussion
recent studies on ncrnas throughout eukaryotes have expanded our understanding of the rna-processing infrastructure  <cit>  with the discovery that key components of the rna-processing machinery occur throughout eukaryotes.
it is now clear that the general ncrna infrastructure has been conserved in excavates, which is an extended but less studied group of eukaryotes.
characterization of the g. intestinalis rnase mrp rna is an important achievement in searching for conserved key ncrnas of the central rna-processing pathway.
sequence and structural analysis of the g. intestinalis mrp rna has shown all the conserved characteristics of eukaryotic mrp, as referred to in figure  <dig>  the conserved structural relationship between g. intestinalis p and mrp rnas indicates that the protein-rna relation in g. intestinalis p and mrp does not differ significantly from other eukaryotes.
identification of the mrp rna from g. intestinalis has filled the gap left from previous studies of mrps.
in looking at the structure of the g. intestinalis mrp we can see how consensus models based on the eukaryotic p <dig> region could not have detected either the sequence or the structure.
the typical p <dig> region of g. intestinalis resembles more the bacterial model of rnase p rna than the eukaryotic p <dig> model for both rnase p and rnase mrp rnas.
however, the rest of the structure fits the eukaryotic rnase mrp rna model.
this demonstrates that with protists in particular, the standard eukaryotic models for ncrnas may not necessarily apply.
the novel h/aca box snornas identified from g. intestinalis and t. vaginalis all have only one predicted target-binding site regardless the number of stem-loops.
having one target-binding site is also seen in the h/aca box snornas found in trypanosomes  <cit> .
identification of the new snornas is usually reliant on predicted conserved target sites, which however appear only partially conserved across distant organisms.
ncrnas in protists may exhibit characteristics not typically seen in more commonly studied model species, and thus these methods may not reveal all the ncrnas within non-model genomes.
for instance the number of pseudouridylation sites in trypanosomes is estimated to be 70- <dig>  <cit> , but to date only around 50% of snornas involved in the modification of these sites have been found  <cit> .
therefore we can expect that the total number of h/aca box snornas will actually be much larger in both g. intestinalis and t. vaginalis.
understanding more about general structural and sequence motifs, will aid us in further searches for h/aca box snornas.
many snornas do not have identified targets and are therefore termed orphan snornas  <cit> .
although there have not been extensive studies, there is no reason to believe that similar orphan snornas will not exist in protists.
a conserved single stem-loop structure constructed by rnamotif  <cit>  was tested to look for possible h/aca-like motifs in g. intestinalis contigs .
results show  <dig> rna contigs adopted the model, and two of them overlap with the snogps-identified candidates.
however, without the constraint of pre-specified target sequences, the predicted structures are not identical.
interestingly, one of the h/aca-like rnas shows a possible target on the g. intestinalis rnase p rna .
however, despite strong g-c rich complementary binding, the potential editing site does not have an un-bound nucleotide immediately 3' to the uridine, as typically observed in all h/aca box snornas found so far.
furthermore, there are only  <dig> base pairs on the 5' side of the base-pairing region instead of  <dig> to  <dig> base pairs typically observed in known snornas.
in addition, there has been no evidence of pseudouridine modification in rnase p. therefore, any conclusion that this ncrna is acting as an orphan snorna on rnasep cannot be made at this stage.
apart from the above, the rest of these  <dig> h/aca-like rnas do not appear to have rrna targets.
we demonstrate in our study that high-throughput sequencing of ncrnas larger than ~21-25nt  is possible, and that the assembly of short read sequences can lead to the characterization of medium-length ncrnas.
we have found this technology to be a boost to the study of ncrnas from non-model eukaryotes and especially those distantly related to well characterized species .
a limitation of this study is that the sequencing length of 36nt  is short and that longer sequences would enable the identification of ncrnas ~70-150nt in single reads, thus not requiring assembly.
CONCLUSIONS
in conclusion, we constructed new rna contigs with updated genomes and identified the rnase mrp of g. intestinalis, which answers positively the previously open question as whether mrp exists in all extant eukaryotes with the a <dig> site.
in addition, a number of new h/aca box snornas have been identified in g. intestinalis and t. vaginalis, with a reduced structure compared to model species, but still possessing the characteristic target-binding pattern and sequence motifs.
it is becoming evident that not only are the components of rna-processing network highly conserved within eukaryotes, but also the pattern of transcription across the genome appears to be shared among distant lineages.
overall, our results imply that it is increasingly likely that the main classes of rna processing and regulation were present in the last common ancestor of eukaryotes  <cit> .
we demonstrate that high-throughput sequencing can be used for the characterization of ncrnas longer than 21-22nt small regulatory rnas for which, in ncrna studies, this technology is typically applied.
our general strategy has been to search for the major classes of rna in all major groups of eukaryotes and investigate the evolution of their mechanisms  <cit> .
increasingly it appears that the major groups are universal in eukaryotes, even though there is continued evolution of individual subgroups of regulatory rna.
discovering how rna systems work in protists, which are distantly remote in an evolutionary sense from other eukaryotes, may hold the key in uncovering how rna mechanisms evolved from our early ancestors.

 the current view of cellular rna organization indicates an rna infrastructure, which describes the spatial and temporal network of the many different rnas. the interconnection of the rna-processing pathways is crucial for cellular processes because different rna processing mechanisms are tightly linked. for example, transcription and splicing happen in close proximity, and splicing is tightly connected with downstream mrna processes including localization, translational regulation, and nonsense-mediated decay. with the advancement of large-scale rna analysis and high-throughput sequencing, conserved features of the eukaryotic rna infrastructure have come to light from studies in animals and plants. in contrast to the genome-wide transcriptional information known in other eukaryotic models, only limited information on rna biology is available from g. intestinalis and t. vaginalis. we can compare the ncrnas from these two protists with other model eukaryotes, to understand more about the general nature of the rna infrastructure within eukaryotes. previous studies in giardia have identified key eukaryotic ncrnas such as the rnase p, snornas, spliceosomal snrnas, mirnas and antisense transcripts. studies on trichomonas ncrnas show that the currently known ncrnas also exhibit typical features of eukaryotes including rnase p, rnase mrp, snrnas and some snornas. however, there are still gaps in our knowledge of rna processing in these species, especially in the characterization of the rnase mrp rna in giardia, and the different types of snornas in trichomonas. annotations of the rnase p rna are also not clear in these genomes. in this study, we characterize medium length ncrnas including rnase mrp, rnase p and snornas from two protozoan parasites: giardia intestinalis and trichomonas vaginalis, to clarify uncertainties about conserved features of the rna infrastructure. we have previously sequenced ncrnas sized from to nucleotides from g. intestinalis and t. vaginalis, and identified from this data small ncrnas such as micrornas in the two protists. using this same sequencing data we are also able to analyze longer ncrnas due to the wide size range. in this study we identify medium-length ncrnas from small rna based sequencing data, characterize the rnase mrp of g. intestinalis, and clarify annotations of rnase p and mrp from both g. intestinalis and t. vaginalis. we also identify new h/aca box snornas from these species. our study clearly demonstrates that high-throughput sequencing cannot only screen for small regulatory rnas, but can also be used for the characterization of longer ncrnas from diverse organisms. results from our work support that g. intestinalis and t. vaginalis possess a rich network of rna processing components expected in the consensus eukaryotic rna infrastructure.  construction of rna contigs using consensus mapping the total rnas from g. intestinalis and t. vaginalis were purified, size fractionated and sequenced according to methods. constructing contigs of short to medium rnas requires a different approach from standard assembly protocols. most ncrnas do not have long poly-a tails; therefore small-rna sequencing is a way of recovering medium-length ncrnas when used in combination with consensus mapping. we successfully generated rna contigs from g. intestinalis and t. vaginalis and compared the updated mapping software bowtie to the original software maq. overall, the contigs constructed using bowtie were longer compared to the ones constructed using maq, although the overall number of contigs was fewer. *mpileup is part of the samtools package with the length cutoff of the new contig datasets set above 50nt we discarded mature mirnas and sirnas. de novo assembly tools such as velvet and abyss produced very few contigs and therefore were not used in this study. hence, we recommend that for medium length ncrna assembly that a reference genome is used for the initial assembly until tools are developed to permit the de novo assembly of small contigs. our study was carried out on g. intestinalis wb strain, and the genome assemblages of the other two strains were used for comparison in some of the subsequent analysis. our rna contigs and trimmed sequences were compared to the available g. intestinalis and t. vaginalis genome annotation to check that rrnas and trnas were represented as expected. from the g. intestinalis sequences all annotated rrnas and trnas from the wb isolate were covered by contigs generated with bowtie mapping. however, many of these contigs contain illumina short reads mapped to multiple sites in the genome and therefore were assigned equally to each of the possibilities. contigs were trimmed in length from the 5' end, to a minimum length of nt, nt and nt to use as sequence tags and mapped against the annotated ncrnas. our original trimmed sequence datasets were also mapped against the annotated ncrnas. all g. intestinalis and t. vaginalis rrnas were found by sequence and contigs. in g. intestinalis all trnas and rrnas were found from assemblage a with trnas not found by contig but found by sequence in assemblage b, and four not found by sequence or contig. a comparison against assemblage e had trnas not found by sequence or contig. given that our sequences came from the same strain as assemblage a, not finding some sequences in the other strains is not surprising. t. vaginalis had many annotations for its rrnas and trnas and the majority were found by both contig and sequence. results from comparing our contigs and sequences with known trnas and rrnas indicate that our method is assembling ncrnas effectively, and that such contigs can be used as sequence tags if trimmed to or nucleotides. identification of the mrp rna from g. intestinalis rnase mrp is a ribonucleoprotein complex, consisting of one ncrna and ~ proteins. it catalyzes the nucleotide cleavage reaction at the a site on the pre-rrna transcript. mrp rna is evolutionarily related to the eukaryotic rnase p rna which processes the 3'- end of pre-trna transcripts. the rnase p and mrp complexes share a number of common protein subunits, and the secondary structures of rnase p and mrp rnas share a common backbone. the rnase p rna has been identified in all eukaryotes and prokaryotes studied to date and shown to be one of the few rnas that have retained catalytic features. the rnase mrp is thought to have evolved before the ancestor of modern eukaryotes, but previously missing evidence of the mrp rna from g. intestinalis imposed a question on the origin of mrp and rrna processing. however the conserved a site on rrna-gene sequence has been identified in g. intestinalis, strongly suggesting the presence of g. intestinalis mrp rna. our contigs contained a sequence that with infernal mapped to the conserved cr- consensus region of the p pseudoknot structure of the mrp core structure. both the pseudoknot and the actual sequences of cr- and cr- regions have previously been shown to be evolutionarily conserved throughout most eukaryotes. sequence alignment of the cr- and downstream predicted cr- regions of g. intestinalis mrp and a representative of other eukaryotic mrps showed a high degree of sequence similarity. upon further analysis, contigs upstream and downstream to the conserved cr- region were found that did not have enough overlap to permit a longer contig to be predicted computationally. this region is highly conserved between the three isolates of g. intestinalis and as expected, our sequence had 100% identity to isolate a. comparing the alignment with the predicted secondary structure indicates that half of the nucleotide differences occur in single stranded regions and all but one change at the beginning of helix p are either compensated with a change at the corresponding position or changed to permit g-u wobble pairing. thus, it is unlikely that any of these changes will have a major impact on the overall rnase mrp rnp secondary structure. all predicted rnase mrp rna and rnase p rnas from g. intestinalis and t. vaginalis are given in table giardia b isolate gs acgj01002297 25811- giardia b isolate gs acgj01002916 916- all genomes downloaded from giardiadb and trichdb the entire predicted secondary structure of g. intestinalis mrp including these conserved regions is shown in figure 3a. a closer look at the structure shows that the cr- region also has the previously identified conserved pattern "anagnna" where the three most conserved "a"s are present. not unexpectedly given the reduced nature of the g. intestinalis genome, the length and structure of the g. intestinalis mrp rna makes it one of the shortest among studied eukaryotes. in t. vaginalis the mrp rna is longer and has a more extended p helix. structurally, the g. intestinalis mrp rna is closer to that of the microsporidium species such as nosema locustae and encephalitozoon cuniculi, which are known to have the shortest mrps. another important characteristic of the mrp rna is the p helix, which is a structure common between rnase mrp and p. the p helix associates with the protein pop, and for mrp it is essential for the cleavage of pre-rrnas at the a site on the pre-ribosomal rna transcript. previous computational studies have already identified both the rnase p and rnase mrp rnas from t. vaginalis. the p helix of g. intestinalis rnase p and rnase mrp are both short and lack the intra-helical loop, however they do share a nt sequence at the terminal loop, consistent with previous studies showing the same relation of p and mrp in a number of eukaryotes. comparison of the p helices of t. vaginalis rnase p and mrp also show a consensus sequence located at the intra-helical loop and the terminal loop regions. the structure of helix p between g. intestinalis mrp and p is also conserved, although no sequence similarity is observed. although we are concentrating on the rnase p and rnase mrp ncrna molecules from g. intestinalis and t. vaginalis, it is worth a quick mention of the important proteins associated with their ribonucleoprotein macromolecules. rnase p and rnase mrp macromolecules share many of their proteins, which have been characterized to small extent in g. intestinalis. some proteins such as the scaffolding protein pop can be hard to identify in protists, due to the large amount of evolutionary distance between them and species from which these proteins are known, but other proteins such as pop are much more conserved across eukaryotes. for a more detailed analysis of the changes in the secondary structure of the rna components we will require further domain and protein structural analysis to understand how the rna and protein components have evolved together. however, even without this detailed protein-rna analysis, we can see that our results strongly support the conservation of the structure-function relationship between rnase mrp and p within g. intestinalis and t. vaginalis. our study also sought to clarify the annotation of the rnase mrp rna especially in the assemblage b from version of the genome. in this annotation within contig acgj the mrp rna is overlapping with a snorna which is in turn overlapping an area annotated as rnase p. the rnase mrp rna from g. intestinalis we have identified does not match this region, but instead this region corresponds to its close relative the rnase p rna. corrected contigs and co-ordinates for the assemblage b genes are given in table although we have characterized the g. intestinalis mrp rna computationally, further molecular biology experimentation will be required before it can be functionally verified. until then, we classify our sequences in g. intestinalis as computationally predicted. new h/aca box snornas small nucleolar rnas are a group of ncrnas of variable length, which are involved in processing of several types of transcripts. most of the known snornas belong to two classes, which are determined by evolutionarily conserved sequence elements: the c/d box and h/aca box. snornas exist in large numbers in eukaryotes. in humans, there have been more than snornas identified, and in general have been shown to have a diverse range of locations and expression patterns. in our previous studies, we have characterized novel c/d box snornas in both g. intestinalis and t. vaginalis. c/d box snornas direct 2'-o methylation, and are relatively easy to identify based on conserved sequence elements and complementary binding to the target rnas. h/aca box snornas direct pseudouridylation, and often exhibit more variable features due to their shorter length of conserved elements and discontinuous complementary target binding regions. as a first step in identification of new h/aca box snornas, the rrnas of g. intestinalis and t. vaginalis were aligned with human rrnas to locate conserved pseudouridylation sites, which were then incorporated into the search for the complementary target-binding regions in candidate h/aca box snornas. after generating the negative controls, the rna contigs were searched using the established parameters from control runs. table shows the scoring rules and results from subsequent analysis. in total, g. intestinalis and t. vaginalis new snornas were identified using this method. the rna sequences and corresponding genomic positions are in additional file and additional file one set of the new g. intestinalis snornas, and two sets of the new t. vaginalis snornas are overlapping in the same genomic region on the plus and minus strand respectively. the structures of the target binding regions of new snornas are shown in figure candidates had either one or two stems, but all contain only one target-binding site. candidates with two stems can have the target-binding site on either stem. further analysis showed that their second stems do not have targets on rrnas despite having the conserved stem-loop structure upstream of the aca box. †the percentage of control sequences with total scores below the cutoff score *number of predicted candidates with total scores above the cutoff score set from control analysis the new g. intestinalis snornas all have different target pseudouridylation sites on rrnas, whereas two of the t. vaginalis new snornas share the same target. in addition, gi/aca. tv/aca. and tv/aca. target a conserved site on the large subunit rrna, the same as gi/aca. and tv/aca. the target regions are highly conserved between g. intestinalis and t. vaginalis, but the sequences of the snornas targeting these sites do not show substantial sequence similarity. a blast search of the newly identified g. intestinalis h/aca box snornas was performed against the genomes of the other two isolates of giardia strains to look for homologous sequences. three g. intestinalis new snornas have homologous sequences in either assemblage b or e, and have homologous sequences in both assemblage b and e. overall the sequences are highly conserved and the nucleotides complementary to the rrna target sequences show minimal changes across the three giardia strains. discussion recent studies on ncrnas throughout eukaryotes have expanded our understanding of the rna-processing infrastructure with the discovery that key components of the rna-processing machinery occur throughout eukaryotes. it is now clear that the general ncrna infrastructure has been conserved in excavates, which is an extended but less studied group of eukaryotes. characterization of the g. intestinalis rnase mrp rna is an important achievement in searching for conserved key ncrnas of the central rna-processing pathway. sequence and structural analysis of the g. intestinalis mrp rna has shown all the conserved characteristics of eukaryotic mrp, as referred to in figure the conserved structural relationship between g. intestinalis p and mrp rnas indicates that the protein-rna relation in g. intestinalis p and mrp does not differ significantly from other eukaryotes. identification of the mrp rna from g. intestinalis has filled the gap left from previous studies of mrps. in looking at the structure of the g. intestinalis mrp we can see how consensus models based on the eukaryotic p region could not have detected either the sequence or the structure. the typical p region of g. intestinalis resembles more the bacterial model of rnase p rna than the eukaryotic p model for both rnase p and rnase mrp rnas. however, the rest of the structure fits the eukaryotic rnase mrp rna model. this demonstrates that with protists in particular, the standard eukaryotic models for ncrnas may not necessarily apply. the novel h/aca box snornas identified from g. intestinalis and t. vaginalis all have only one predicted target-binding site regardless the number of stem-loops. having one target-binding site is also seen in the h/aca box snornas found in trypanosomes. identification of the new snornas is usually reliant on predicted conserved target sites, which however appear only partially conserved across distant organisms. ncrnas in protists may exhibit characteristics not typically seen in more commonly studied model species, and thus these methods may not reveal all the ncrnas within non-model genomes. for instance the number of pseudouridylation sites in trypanosomes is estimated to be 70-, but to date only around 50% of snornas involved in the modification of these sites have been found. therefore we can expect that the total number of h/aca box snornas will actually be much larger in both g. intestinalis and t. vaginalis. understanding more about general structural and sequence motifs, will aid us in further searches for h/aca box snornas. many snornas do not have identified targets and are therefore termed orphan snornas. although there have not been extensive studies, there is no reason to believe that similar orphan snornas will not exist in protists. a conserved single stem-loop structure constructed by rnamotif was tested to look for possible h/aca-like motifs in g. intestinalis contigs. results show rna contigs adopted the model, and two of them overlap with the snogps-identified candidates. however, without the constraint of pre-specified target sequences, the predicted structures are not identical. interestingly, one of the h/aca-like rnas shows a possible target on the g. intestinalis rnase p rna. however, despite strong g-c rich complementary binding, the potential editing site does not have an un-bound nucleotide immediately 3' to the uridine, as typically observed in all h/aca box snornas found so far. furthermore, there are only base pairs on the 5' side of the base-pairing region instead of to base pairs typically observed in known snornas. in addition, there has been no evidence of pseudouridine modification in rnase p. therefore, any conclusion that this ncrna is acting as an orphan snorna on rnasep cannot be made at this stage. apart from the above, the rest of these h/aca-like rnas do not appear to have rrna targets. we demonstrate in our study that high-throughput sequencing of ncrnas larger than ~21-25nt is possible, and that the assembly of short read sequences can lead to the characterization of medium-length ncrnas. we have found this technology to be a boost to the study of ncrnas from non-model eukaryotes and especially those distantly related to well characterized species. a limitation of this study is that the sequencing length of 36nt is short and that longer sequences would enable the identification of ncrnas ~70-150nt in single reads, thus not requiring assembly.  in conclusion, we constructed new rna contigs with updated genomes and identified the rnase mrp of g. intestinalis, which answers positively the previously open question as whether mrp exists in all extant eukaryotes with the a site. in addition, a number of new h/aca box snornas have been identified in g. intestinalis and t. vaginalis, with a reduced structure compared to model species, but still possessing the characteristic target-binding pattern and sequence motifs. it is becoming evident that not only are the components of rna-processing network highly conserved within eukaryotes, but also the pattern of transcription across the genome appears to be shared among distant lineages. overall, our results imply that it is increasingly likely that the main classes of rna processing and regulation were present in the last common ancestor of eukaryotes. we demonstrate that high-throughput sequencing can be used for the characterization of ncrnas longer than 21-22nt small regulatory rnas for which, in ncrna studies, this technology is typically applied. our general strategy has been to search for the major classes of rna in all major groups of eukaryotes and investigate the evolution of their mechanisms. increasingly it appears that the major groups are universal in eukaryotes, even though there is continued evolution of individual subgroups of regulatory rna. discovering how rna systems work in protists, which are distantly remote in an evolutionary sense from other eukaryotes, may hold the key in uncovering how rna mechanisms evolved from our early ancestors. 
in this study we have conducted a genome-scale survey of medium-length ncrnas from the protozoan parasites giardia intestinalis and trichomonas vaginalis. results indicate that giardia intestinalis and trichomonas vaginalis, like their distant multicellular relatives, contain a rich infrastructure of rna-based processing. we have also uncovered new h/aca box snornas, expanding our knowledge of the h/aca family of snornas.
 eukaryotic cells possess a complex network of rna machineries which function in rna-processing and cellular regulation which includes transcription, translation, silencing, editing and epigenetic control. studies of model organisms have shown that many ncrnas of the rna-infrastructure are highly conserved, but little is known from non-model protists. in this study we have conducted a genome-scale survey of medium-length ncrnas from the protozoan parasites giardia intestinalis and trichomonas vaginalis.  we have identified the previously'missing' giardia rnase mrp rna, which is a key ribozyme involved in pre-rrna processing. we have also uncovered new h/aca box snornas, expanding our knowledge of the h/aca family of snornas.  results indicate that giardia intestinalis and trichomonas vaginalis, like their distant multicellular relatives, contain a rich infrastructure of rna-based processing. from here we can investigate the evolution of rna processing networks in eukaryotes. 