BACKGROUND
rna interference  is a well-conserved gene regulatory pathway found in most
eukaryotes  <cit> .
many important biological functions are controlled by rnai such as
developmental regulation  <cit> , genome protection against viruses and transposons  <cit> , and dna elimination  <cit> .
small rna  molecules, usually  <dig> – 30nt, are the key
elements for rnai.
guided by their associated protein complexes, they base-pair to
the targeted transcripts or genomic loci to trigger gene silencing at either the
transcriptional or post-transcriptional level  <cit> .
in recent years, high throughput sequencing has facilitated the
identification of diverse species of small rnas in different organisms  <cit> .
several protozoan parasites such as trypanosoma brucei, toxoplasma gondii,
giardia lamblia, trichomonas vaginalis, and entamoeba
histolytica contain key genes of the rnai pathway in their genomes  <cit> .
the functions of rnai in parasite biology include retrotransposon
control in t. brucei <cit> , gene regulation in e. histolytica <cit> , and control of antigenic variation in g. lamblia <cit> .
in t. gondii and t. vaginalis the studies have been
limited to endogenous small rna sequencing with no functional studies yet reported  <cit> .
e. histolytica causes dysentery and liver abscesses in humans and affects
<dig> million people worldwide  <cit> .
the study of this important human parasite has been hampered by lack of
standard molecular genetic tools due to the polyploid nature of the e.
histolytica genome  <cit> .
recently, several rnai-based gene knockdown approaches -- dsrna/sirna  <cit> , short-hairpin rna  <cit>  and a transcriptional gene silencing approach in the g <dig> parasite strain  <cit>  -- have been established in this organism.
we have shown that e.
histolytica has a 27nt small rna population, which has
5′-polyphosphate  and
3′-oh termini and associates with ehago2- <dig>  <cit> .
additionally, we have demonstrated that gene silencing in the e.
histolytica g <dig> strain is mediated through a sirna pathway  <cit> .
the e. histolytica genome encodes three argonaute proteins  of which ehago2- <dig>  is highly expressed and
associates with 27nt small rnas  <cit> .
in this report, we immunoprecipitated small rnas bound to ehago2- <dig> and
sequenced them using a high throughput pyrosequencing approach, which generated over
<dig>  small rna reads.
analysis of these endogenous small rnas revealed that their
peak-length is 27nt and that there was a strong g bias at the
5′-nucleotide.
genome analysis showed that small rnas largely
mapped to annotated protein coding genes , which can be categorized into three groups:  genes with
only antisense small rnas;  genes with both antisense and sense small rnas; and
genes with only sense small rnas.
biochemical analysis revealed that both
sense and antisense small rnas  have
5′-polyp termini.
strand specific rt-pcr showed both sense and
antisense transcripts can be detected for group ii gene loci, suggesting that
bi-directional transcripts are available to serve as templates for small rna
generation.
identification of small rnas that map to introns as well as exon-exon
junctions indicated that e. histolytica rna-dependent rna polymerase
could use both nascent and mature transcript as a template for generating small
rnas.
whole genome microarray data indicated that most protein coding genes with
large numbers of antisense small rnas are not expressed, indicating that antisense
small rnas likely play a role in gene silencing.
we further sequenced a
size-fractionated small rna library from the non-virulent e. histolytica
rahman strain and identified strain-specific patterns of antisense small rnas
mapping to a virulence gene in a manner consistent with its regulation by small
rnas.
in summary, we have characterized the endogenous small rna populations from a
virulent and non-virulent strain of e. histolytica using a high-throughput
small rna pyrosequencing approach.
our analysis indicates that the small rna pathway
likely regulates expression of strain-specific genes in this parasite system,
including some virulence determinants.
RESULTS
high-throughput pyrophosphate sequencing of ehago2-2-bound small rnas
a library of ehago2- <dig> associated small rnas was generated from rna obtained by
immunoprecipitation  with anti-myc antibody in e. histolytica
hm-1:imss cells stably expressing myc-tagged ehago2- <dig>  we have previously shown
that ehago2- <dig> specifically associates with 27nt small rnas, which have
5′-polyp and 3′-oh termini  <cit> .
thus, the small rna library was generated using a
5′-p independent cloning approach .
<dig> genome sequencer process-specific a and b adaptors
were incorporated into the cdna small rna library by pcr and a full sequencing
run was performed generating a total of  <dig>  sequences.
using the small rna
sequence analysis workflow , we removed adaptor and linker sequences and limited the size to 15-40nt.
the resulting output contained  <dig>  sequences that represented  <dig>  unique
sequence reads.
about  <dig> %  of the unique sequences were cloned only
once indicating that the current sequencing was far from saturating
.
library from e. histolytica hm-1:imss
5′-p independent cloning
one pyrophosphate sequencing run was performed.
unix tools were used
to remove adaptor and linker sequences.
the bowtie alignment tool
was used to scan for structural rnas, repetitive elements and for
the final genome mapping.
the numbers of reads that map to the
genome sequence at each step are listed.
we used the bowtie alignment tool  <cit>  to filter out reads that mapped to structural rnas: trna , rrna  and repetitive elements  .
the remaining dataset  was mapped to the
e. histolytica hm-1:imss genomic sequence.
a total of  <dig>  reads
mapped to the genome  with
<dig>  reads mapping to open reading frames and  <dig>  reads mapping to a
single gene locus .
this
dataset targets ~4% of annotated protein coding genes in the e.
histolytica genome .
the genome mapping revealed some striking features.
first, many small rna reads mapped antisense to annotated protein coding genes
; small rnas that mapped to intergenic regions  and sense to
protein coding genes  were less common .
second, small rnas mapping to sine/line retrotransposon
elements and other potential repetitive regions in the genome only accounted for
5% and  <dig> % of the reads respectively.
this demonstrates that ehago2- <dig> is not
primarily associated with small rnas derived from transposons, in contrast to
the single t. brucei argonaute protein, which has been shown to
associate with small rnas that map primarily to transposons and are thought to
control their expression  <cit> .
third, the mapping of small rna to the genome indicated that small
rna reads tend to be derived from a small number of genomic locations or
“hot spots”.
when the genome was scanned using a 500 bp window,
the majority of small rnas arose from  <dig> % of total windows in genome.
this
suggests that the small rnas could associate with certain genomic features such
as repeat regions, centromeric or telomeric regions, which have been shown to be
a source of small rnas in other systems  <cit> .
in summary, our pyrosequencing data indicated that ehago2-2
associates with an abundant endogenous small rna population, which is largely
derived from the predicted protein coding genes in e. histolytica.
e. histolytica hm-1:imss trophozoites.
the composition of small rnas from the ehago2- <dig> ip
small rna library that match the e. histolytica genome
annotation in the hm-1:imss strain.
small rna length
distribution for all reads  and all reads remaining
after filtering out those that originate from structural rnas and
repetitive elements reads  is shown.
nucleotide frequency at each position for the aligned 27nt reads reveals
a 5′-g predominance
small rnas are largely 27nt size with a striking 5′-g
bias
we analyzed the size distribution and nucleotide composition of small rnas and
found that all reads
peaked sharply at 27nt , consistent with
the size of small rnas previously noted to associate with ehago2- <dig>  <cit> .
when nucleotide frequency was plotted at each position for the 27nt
population we noticed a striking 5′-g bias .
the nucleotide frequency for the 26nt and 28nt small
rnas also shows a 5′-g propensity, but this was not the case
for 17nt small rnas indicating that the smaller sequences are likely degradation
products .
given that
the e. histolytica genome is very at-rich   <cit> , the 5′-g bias in small rnas is remarkable when
compared to all the remaining plotted positions for small rnas.
a
5′-g bias in small rnas has been reported in two other
organisms, i.e.
c. elegans and ascaris, where 22g populations
with 5′-polyp termini are defined as secondary sirnas and
thought to be generated by rdrp  <cit> .
thus, we reason that the 5′-g biased 27nt small
rnas are likely rdrp-related and function as silencing sirnas in e.
histolytica.
small rna distribution patterns in the genome
the e. histolytica genome was first published in  <dig>  <cit>  and a second version including new assemblies and reannotation was
released in  <dig>  <cit> .
the current genome assembly is still in scaffold stage, which
contains  <dig>  supercontigs and  <dig>  genes.
to gain an overview of small rna
distribution in the genome, we mapped small rnas using the bowtie alignment tool  <cit> .
we identified  <dig> supercontigs that had ≥ <dig> small rnas, and
which accounted for 52% of the genome size  and which
contained 96% of the total small rnas.
specifically, the small rnas were highly
enriched on  <dig> supercontigs that although only  <dig>  mb in total size
contained ~50% of all sequenced small rnas .
as the structure of the genome is unknown at
present, we do not know if the  <dig> supercontigs that were enriched in small rnas
belong to centromeric or telomeric regions.
to analyze the overall small rna
density distribution on the genome, we scanned the genome using a 500 bp
window, and counted numbers of small rnas in each window.
we defined a
“hot spot” as containing ≥ <dig> small rnas per window; using
these parameters, there were  <dig> “hot spot” windows out of  <dig>
genomic windows.
the graphic views of “hot spot” distribution
further revealed that most small rnas arose either from some large clusters
or from isolated peaks .
there are regions of the genome
that had only a few mapped small rnas .
we analyzed the protein coding genes to which small rnas mapped, and found that
many genes had only a few small rnas that mapped to them and hence are likely
artifacts.
thus, we tested our dataset with different cutoffs for the number of
small rnas mapping to a gene; we used four measures .
for each cutoff, we
identified the number of protein coding genes in each category  and
plotted the microarray expression value for these genes .
for the two least stringent criteria , we observed no significant difference
in the microarray expression value for the three categories of protein coding
genes .
however, when we used either the
≥ <dig> or ≥ <dig> small rna cutoff, we identified significantly lower
expression among genes with antisense or sense/antisense small rnas compared to
genes with sense small rnas .
the total
number of protein coding genes using either cutoff  was relatively similar .
to be as
stringent as possible, we decided to use a cutoff of ≥ <dig> small rnas
mapping to a gene for further analysis.
overall,  <dig> protein coding genes  had ≥ <dig> small rnas that mapped to them.
these protein
coding genes could be categorized into three groups:   <dig> genes with only
antisense small rnas;   <dig> genes with both antisense and sense small rnas;
and   <dig> genes with only sense small rnas.
most genes in group i and ii are
annotated as hypothetical proteins .
however, a few gene
families were represented including aig <dig> family proteins ,
beta-amylase , deoxyuridine 5′-triphosphate
nucleotidohydrolase domain proteins , dna polymerase , and c2
domain proteins .
in order to determine whether protein coding genes with small rnas are in
proximity to each other, we characterized the patterns of genes to which small
rnas map.
a cluster is defined as ≥ <dig> contiguous genes .
a pair is defined as  <dig> contiguous genes  that are ≤1000 bp apart.
there are a total of  <dig> protein coding genes that have ≥ <dig> small rnas and
of these the majority are in clusters  or in pairs .
these
clustered/paired genes were largely in group i and ii categories.
we next looked
at transcript orientation in the paired genes.
the  <dig> protein coding genes in
pairs had  <dig> divergent pairs ,  <dig> convergent pairs
, and  <dig> tandem pairs .
a total of  <dig> protein coding genes are unpaired with 63
genes in group i,  <dig> genes in group ii, and  <dig> genes in group iii.
when we characterized clustered protein coding genes  with
small rnas, we identified  <dig> clusters, which ranged in size from a cluster of 3
genes to clusters as large as  <dig> genes .
a substantial number  of clusters are
in previously identified regions of d <dig>  d <dig> and d <dig> genome duplication, which are
segmental duplications with each segment flanked on both ends by inverted
repeats such as ir/ehere1/ehlines  <cit> .
clusters that are not in regions of d1-d <dig> genome duplications are
still flanked by repetitive elements at either one or both ends .
thus, clustered genes  are more likely to be associated with repetitive
elements.
in order to determine whether small rnas are enriched on paired or clustered
protein coding genes or in the intergenic dna regions, we calculated the small
rna density on these paired/clustered genes as well as the intergenic regions
between genes.
this was calculated as small rna/bp.
we identified that the
density of small rnas mapping to intergenic regions was significantly lower
compared to small rna density mapping to paired/clustered genes .
this indicates that small rna synthesis is
most likely templated using a given gene rather than a long template covering
several genes.
for intergenic regions that had high small rna density, we found
these small rnas are often in discrete sections or adjacent to predicted genes.
thus, we postulate that this may be due to small rnas mapping to an unannotated
gene or utrs.
in summary, our analysis suggests that the small rna targeted protein coding
genes tend to be in pairs or clusters, and that clusters of genes with small
rnas are more often associated with repetitive elements.
small rna density on
paired/clustered genes versus intergenic regions implies that it is unlikely for
either dna or a long transcript covering several genes to be used as a template,
but rather that transcript derived from each gene is the most likely
template.
small rna distribution patterns within protein coding genes
we have previously shown that small rnas that map antisense to protein coding
genes tend to be most abundant toward the 5′-end of genes  <cit> .
however, that analysis was done using a very limited dataset of
small rnas generated from sanger sequencing.
our new pyrophosphate sequencing
dataset enabled us to examine this observation on a larger scale.
using the
stringent criteria of ≥ <dig> small rnas mapping to a gene, a total of 226
protein coding genes were categorized as group i .
we plotted small rna distribution along each gene  .
there was a clear trend showing that most antisense small rnas
mapped toward the 5′-termini of predicted genes.
this trend
holds true for most targeted protein coding genes  and was not
caused by a few genes with a high number of small rnas at the
5′-end.
for the 5′-polyp small rnas in
ascaris and c. elegans, there is a clear difference of
small rna distribution on their corresponding mrna targets.
in c.
elegans 22g-rnas are mostly enriched at 3′-end of the
mrna  <cit> , while ascaris 22g-rnas are distributed toward the
5′-end of mrnas  <cit> .
our results indicate that the small rna distribution pattern for the
antisense small rnas in e. histolytica is more similar to that in
ascaris as compared to c. elegans.
the
5′-bias of antisense small rnas could reflect the heavy
recruitment of rdrp complexes to these regions, and the exact mechanism of rdrp
in generating secondary antisense small rnas is largely unknown at present.
protein coding genes.
each small rna read was assigned a
position value based on the position of the starting nucleotide along
the gene.
the gene length for each protein coding gene was normalized to
one.
total number of small rna reads  was plotted from
5′ to 3′ according to their
relative position within all genes .
small rna
distribution on genes with only antisense small rnas.
small
rna distribution on genes with both antisense  and sense small
rnas.
small rna distribution on genes with only sense
small rnas.
grey bars: antisense small rnas; white bars: sense small
rnas.
for the group ii genes , we similarly plotted the small rna distribution along each gene.
we
noted that antisense small rnas were distributed with a
5′-enrichment , while the sense small rna
distribution pattern was more heterogeneous  .
additionally, we noted that
for group ii genes, in most cases the number of antisense small rnas was greater
than the number of sense small rnas for each gene locus .
genes with both antisense and sense small rnas.
ehi_ <dig>  is depicted with the small rnas that
map to it; each bar represents one unique small rna .
the positions of selected probes used
for small rna northern blot analysis are represented by bars and numbers
.
the positions of the f and r primers, used to generate cdna for
the strand-specific rt-pcr, are shown.
northern blot analysis
detected signal for antisense small rnas  and sense
small rnas .
sense small rnas have
5′-polyp termini.
10 μg small rna
enriched sample was treated with terminator or capping enzyme.
probes  <dig>
<dig>   <dig> were used for northern blot analysis.
a control oligonucleotide
that is labeled with a 5′-monop is degraded by
terminator and has no change in size with capping enzyme, as expected.
strand specific rt-pcr demonstrates both sense and
antisense transcripts for ehi_ <dig>  cdna was generated using f and r
primer  as well
as oligo dt primer.
rt-pcr reveals both antisense and sense transcripts
with antisense transcript at lower abundance than sense transcript.
both
rt  and control reactions lacking rt  are shown.
in contrast, when small rna distribution was plotted for group iii genes , it became apparent that sense small rnas were
enriched towards the 3′-end of each gene
.
we noticed that genes with only
sense small rnas mainly code for four highly expressed gene families ; gal/galnac lectin ; three protein
kinase families ; and hypothetical proteins ).
since our
cloning method will capture all types of small rnas ,
degradation products with 5′-oh species would also be cloned;
this may suggest that at least some of the sense small rnas might be mrna
degradation products.
however, we found no sense small rnas to other highly
expressed genes, indicating some specificity for sense small rnas to these
specific loci.
since the mapping of sense small rnas to many genes often had
abrupt boundaries within/near 3′ end of genes and sometimes
extended into intergenic regions , we felt that some mapping artifacts could be due to poor genome
quality in these regions.
as the e. histolytica genome is not complete
at present, we cannot rule out whether or not these sense small rnas are derived
from some other loci.
although sense small rnas have been identified in almost all cloning libraries,
these types of small rnas have largely been ignored since it is hard to evaluate
their identity as true endogenous small rnas versus non-specific hydrolysis
fragments.
in both c. elegans and ascaris suum, where
5′-polyp small rnas were found, sense small rnas were
identified but were not characterized  <cit> .
for the e. histolytica group iii genes that have only sense
small rnas mapped to them we have not made any assumptions and have not further
characterized the structure, derivation, or function of this category of sense
small rnas.
antisense and sense small rnas  have
5′-polyphosphate termini
the secondary 5′-polyp small rnas in nematodes  are thought to be generated by an
amplified gene silencing mechanism, likely through rdrp  <cit> .
we have previously demonstrated that in e. histolytica both
endogenous and exogenously triggered antisense small rnas have
5′-polyp termini  <cit> .
since our analysis to date had focused on the antisense small rnas,
we wanted to determine whether the small rnas that map sense to genes  also have 5′-polyp termini.
for this
purpose, we analyzed small rnas that mapped sense to the ehi_ <dig> locus.
this
gene is highly enriched for both antisense and sense small rnas with small rna
coverage extending into the apparent 5′-upstream region
.
northern blot analysis detected
signal at 27nt with probes that detect antisense small rnas
and with probes that detect sense small rnas  .
all the blots were performed using the same membrane,
and therefore the relative band intensity reflects the abundance of small rnas.
we observed a correlation of 5'-enriched distribution of small rnas with the
intensity of probe 1 > probe 2 > probe  <dig>  we then
performed a terminator exonuclease assay and a 5′-end capping
assay on the total rna sample.
the signal for sense small rnas
were resistant to terminator treatment and shifted up after capping assay,
indicating that these small rnas that map sense to the ehi_ <dig> locus have
5′-polyp termini .
probe  <dig>  which would detect antisense small rnas had the expected biochemical
features consistent with 5′-polyp termini.
the control  was, as expected, degraded by
terminator enzyme and unaffected by treatment with capping enzyme.
we further examined a second example: the locus
that contains both antisense and sense small
rnas to ehi_ <dig> and ehi_ <dig> as well as one potential unannotated gene
.
probes for detecting
antisense  and sense  small rnas were chosen for ehi_ <dig>
northern blot analysis showed signal for both probes at ~27nt; the sense small
rna was resistant to terminator exonuclease and thus has
5′-polyphosphate termini.
thus, for the two loci tested  we determined that the
sense small rnas also have 5′-polyp termini.
the biochemical features of sense small rnas  having
5′-polyp termini was unexpected.
analysis for pairing
between antisense and sense small rnas showed no enriched pairs for these gene
loci .
in a typical rnai pathway, dsrna is chopped into a sirna
duplex, where enriched pairing between antisense and sense small rnas can be
found  <cit> .
the lack of pairing between antisense and sense small rnas in group
ii genes and the 5′-polyp termini for both antisense and sense
small rnas indicates that these small rnas could be individually processed from
bidirectional transcripts at these loci.
to determine if this may have occurred,
we performed strand-specific rt-pcr for ehi_ <dig>  ehi_ <dig> and ehi_ <dig>
at all three loci, both sense and antisense transcripts can be detected, albeit
with antisense transcripts at much lower level than the sense transcript
.
overall transcript levels as assayed by rt-pcr
correlated with the abundance of antisense and sense small rnas mapped to these
loci.
we further applied the strand-specific rt-pcr assay to two additional
protein coding genes , which had both antisense and
sense small rnas and identified transcripts in both directions for these loci
.
thus, we conclude that although bidirectional transcription
has not been previously documented in e. histolytica, both antisense
and sense small rnas are likely templated from bidirectional transcripts and
generated by an rdrp-dependent mechanism resulting in small rnas with
5′-polyphosphate termini.
natural antisense transcripts
have been shown to be a major source of sirna generation in drosophila
melanogaster <cit>  and in plants  <cit> .
in the parasite, giardia lamblia, bidirectional
transcription produces abundant sterile antisense transcripts  <cit> .
further studies to characterize the extent of natural antisense
transcripts in e. histolytica and the mechanism for generation of
5′-polyp small rnas are needed.
our demonstration that
small rnas to group ii genes
have 5′-polyp termini and strand-specific rt-pcr detecting
transcripts in both directions indicates that these sense small rnas are likely
generated from antisense transcript, and indicates that some portion of
“sense” small rnas in our dataset although called
“sense”, are truly antisense to the transcript derived from the
opposite strand of the examined gene.
small rnas are derived from both unspliced and spliced transcripts
in order to identify whether small rnas map to spliced or unspliced transcripts,
we downloaded both genomic and mrna sequences for all e. histolytica
protein coding genes with at least one predicted intron.
small rnas mapping to
introns are defined as those that map to the genomic gene sequence but not the
mrna sequence.
small rnas mapping to exon-exon junctions are those that map to
mrna sequence but not the genomic gene sequence.
we found a total of  <dig> small
rna reads that spanned exon-exon junctions  and  <dig>  small rna reads
that mapped to predicted introns  .
we further examined the protein coding genes with at least  <dig> small rnas and
which also have at least one intron .
for this list, we first checked for potential
“false introns” caused by genome sequence error, and excluded
ehi_ <dig>  ehi_ <dig> and ehi_ <dig> as they have many ns in their intron
sequences.
we then checked the predicted intronic sequence in the remaining
genes for an in-frame stop codon or frame disruption, as this strongly suggests
that the intron is correctly predicted .
lastly, we checked for paralogs within these genes.
with these criteria, we examined the three categories of genes to which small
rnas mapped to identify small rnas that mapped to exons, introns and exon-exon
junctions .
for the group
i genes,  <dig> unique genes had small rnas that mapped to predicted introns and
among them,  <dig> unique genes also had small rnas that mapped to exon-exon
junctions.
for the group ii genes,  <dig> unique genes had antisense small rnas that
mapped to introns and  <dig> of these also had sense small rnas that mapped to
introns.
none of the genes in this category had small rnas that mapped to
exon-exon junctions.
for the group iii genes,  <dig> unique genes had small rnas
mapped to predicted introns, but none mapped to exon-exon junctions.
overall, we
made a number of observations:  a greater number of small rnas mapped to
exons than to the introns for all three groups of genes;  all but one
intron-containing gene in groups i and ii had small rnas that mapped to introns;
and  only a limited number of genes  had small rnas that
mapped to exon-exon junctions.
these data indicate that both spliced and
unspliced transcripts are capable of being used as templates to produce small
rnas in e. histolytica.
as an example, the mapping of small rnas to
exons, introns and exon-exon junctions are shown for ehi_ <dig> and ehi_197360
genes .
further
calculations of the small rna density revealed four-fold greater density of
small rnas in exons than in introns [ehi_ <dig>  exon  vs. intron ;
ehi_ <dig>  exon  vs. intron ] .
the difference could suggest that spliced
transcripts are preferred as templates to unspliced transcripts, or
alternatively may simply be a reflection of the ratio of spliced and unspliced
transcripts available in the cell.
in c. elegans, ego- <dig>  an rdrp, is critical for c. elegans
germline development and is responsible for producing 5′-polyp
antisense small rnas from mrna-derived loci.
small rna sequencing has shown that
small rnas often span exon-exon junctions and rarely map to introns, indicating
ego- <dig> uses processed mrna as a template  <cit> .
rdrp could theoretically template on genomic dna, nascent
transcripts, or processed mrnas.
small rnas that map to exon-exon junctions
provide evidence that a spliced mrna template is used to generate these small
rnas, whereas small rnas that map to introns indicate that non-spliced templates
can also be used to generate small rnas.
based on the observations that there
are small rna free genomic regions between genes with antisense small rnas, and
that many more small rnas map to exons than introns, we conclude that the e.
histolytica rnai machinery prefers mature transcript as a template for
generating small rnas.
however, the machinery in e. histolytica also
seems capable of using unspliced transcripts as template, although at reduced
levels.
whether this is indicative of the inherent preference of the e.
histolytica machinery or due instead to the low abundance of unspliced
mrna is not clear at present.
small rnas that map to trnas, rrnas and retrotransposon elements
in order to identify small rnas that map to the trnas, rrnas and retrotransposon
elements, we followed the outline in additional file 1: figure s <dig>  e. histolytica has uniquely organized trna
genes that are in multiple tandem-array units, likely arranged at subtelomeric
regions and spaced by tandem repeats of at-rich sequences  <cit> .
the e. histolytica rrna genes reside on an extrachromosomal
circular plasmid and two rrna transcription units are organized as inverted
repeats  <cit> .
we mapped the small rna reads to the trna repeat units and the rrna
plasmid.
we found that nearly all small rna reads were in the sense orientation
to the coded trnas and rrnas.
additionally, we plotted the size distribution and
nucleotide frequency for rrna reads and noted that small rnas in these
categories did not peak at 27nt , and did not have a 5′-g enrichment  indicating that these small rnas
are most likely degradation products resulting from these highly expressed
structural rnas.
however, we are aware of some recent reports showing that small
rnas could originate from trnas and snornas  <cit> .
upon a closer examination of the trna reads, we noticed a slight
peak at 27nt with some degree of 5′-g enrichment for this 27nt
population only .
whether or not this
indicates that these are functional small rnas in entamoeba needs
further study.
transposons and repetitive dna are abundant in entamoeba and hundreds of
copies of the long interspersed nuclear elements  and short interspersed
nuclear elements  can be found in the e. histolytica genome  <cit> .
our small rna dataset contains 5% of reads that mapped to line and
sine elements.
analysis of the lengths of these small rnas showed two peaks  .
when nucleotide composition is plotted, the 5′-g
propensity is apparent for the 27nt peak, but not for the 17nt peak .
thus, the 27nt small rna
population that maps to line/sine elements had features similar to those that
map to coding regions and are likely not artifacts.
as an example, we mapped
small rnas to the ehrle <dig> sequence, which has been categorized in the ehline1
family  <cit> .
the small rnas are scattered along the whole region on both strands
and cover the whole ehrle unit, with a slight increase in small rnas near each
end .
we got a positive
signal from northern blot analysis using several probes to retrotransposon
elements although the size by northern blot analysis was slightly higher
than the cloned small rna.
this indicates that small rnas could derive from
these retrotransposon elements.
the mapping of small rnas to d1-d <dig> repetitive segments showed a large number of
small rna reads on d <dig>  d <dig> and d <dig> segments but not on d <dig> segments .
annotated protein coding genes in
these duplication regions appear to be covered by large numbers of antisense
small rnas, forming a large cluster .
thus, although the overall numbers of small rnas that associate with
ehago2- <dig> and map to repeat or retrotransposon elements is low, they may play a
functional role in controlling genome stability as has been shown in other
systems  <cit> .
alternatively, small rnas may have a role in controlling these
retrotransposon elements but may do so by associating with the two other
argonaute proteins in e. histolytica.
a global assessment of genes potentially regulated by small rnas in e.
histolytica
we have previously shown an inverse correlation between gene expression and
antisense small rna abundance raising the intriguing possibility that antisense
small rnas may mediate target gene silencing in e. histolytica <cit> .
however, those data were on a very limited scale due to the very
limited set of sequenced small rnas.
the pyrosequencing dataset allowed us to
assess the potential genome-wide affects of these small rnas by comparing small
rna abundance with microarray expression data.
we used previously published
microarray data from e. histolytica hm-1:imss trophozoites   <cit> .
the analysis was conducted for the three groups of protein coding
genes with distinct small rna mapping patterns.
for group i genes , there were  <dig> protein coding
genes that met the criteria; of these,  <dig> genes are represented on the
microarray.
we plotted the number of mapped small rna reads for each gene as a
function of normalized microarray expression data and identified that most genes
in this category are not expressed .
for group ii genes  there are  <dig> genes that met our criteria
and  <dig> are represented on the microarray and most of these genes  are also
not expressed .
in order to determine whether these  <dig> genes  are expressed under other conditions, we
compared the expression profiles of these genes across all other conditions
tested   <cit> .
of these  <dig> genes,  <dig> were expressed in a strain-specific manner,
<dig> were regulated under various culture conditions, and  <dig> were regulated during
stage conversion  .
overall, a total of  <dig> protein
coding genes are not expressed in e. histolytica hm-1:imss trophozoites
under standard in vitro growth conditions  <cit> .
based on our sequencing data we estimated that small rnas target
about 41% of these protein coding genes .
of these 112
genes, a large proportion  change in expression profile under one or
more conditions examined indicating that they are not permanently silenced and
may be regulated by small rnas.
of small rnas targeting to a gene.
genes are plotted using the
number of mapped small rnas  and their normalized microarray
expression levels .
the dashed line indicates the cutoff
for gene expression generally detectable by rt-pcr.
plot for
group i genes .
plot for group ii genes .
plot for group iii genes .
box-and-whisker expression plots for genes in groups i, ii, iii as well
as all genes represented on the microarray.
the top and bottom ends of
each box represent the 75th and 25th percentile, respectively; the
middle line represents the median value.
for the genes in group iii ,  <dig> genes met our
criteria with  <dig> represented on the microarray.
plotting the number of mapped
reads for each gene as a function of its normalized microarray gene expression
value showed no direct link between numbers of sense small rnas to a gene and
the expression level of that gene .
we reasoned that for genes with high expression
value, the sense small rnas may represent degradation products.
however, for
genes with very low mrna expression , we have no
good explanation on how these sense small rnas were generated.
whether this
represents an artifact of errors in genome assembly or some other factor is not
clear at present.
interestingly, none of these genes are regulated under other
conditions tested .
in summary, the strongest correlation between gene expression and small rna
abundance was for genes that had either abundant antisense small rnas or
abundant sense and antisense small rnas.
although simply correlative at present,
the inverse correlation between antisense small rna abundance and gene
expression suggests that antisense small rnas mediate target gene silencing in
e. histolytica and are potentially involved in gene regulation
under various conditions.
small rna sequencing from the nonvirulent e. histolytica rahman
strain
it has been observed that pathogenicity varies greatly among different e.
histolytica strains.
e. histolytica hm-1:imss is a virulent
strain, while e. histolytica rahman is a nonvirulent strain  <cit> .
we have previously demonstrated that gene expression profiles are
substantially different between these two strains and have used the distinct
strain-specific expression profiles to identify virulence genes  <cit> .
given the data above, and to explore whether small rnas play a role
in strain-specific and/or virulence gene regulation, we constructed a small rna
library from trophozoites of the e. histolytica rahman strain.
we visualized the small rna populations in e. histolytica rahman by
separating total rna on a 12% denaturing polyacrylamide gel followed by sybr
gold staining and visualized an abundant 27nt small rna population .
a small rna library was constructed from size-fractionated rna
using a 5′-p independent cloning approach and limited
pyrophosphate sequencing was performed generating  <dig>  reads.
for the purpose
of mapping, we used e. histolytica hm-1:imss genome as a reference
genome rather than the current e. histolytica rahman assembly, based on
the following facts:  the current e. histolytica rahman genome
assembly  is in a preliminary stage
containing  <dig>  small contigs and is unannotated,  there is a high
similarity between these two strains and one previous study has estimated that
only  <dig> out of a sample of  <dig>   genes were identified as highly or
significantly divergent  <cit> , and  affymetrix platform microarrays found no difference in
overall hybridization efficiency levels compared to hm-1:imss , indicating a high level of sequence identity for
the protein coding genes  <cit> .
we realize that sequence differences between the e.
histolytica hm-1:imss and rahman strains may cause us to lose some
data.
however, the advantages of being able to map to an annotated genome and
thus determine how many small rnas map to protein coding genes  and to intergenic regions were significant enough that
we proceeded with the data generated by aligning the e. histolytica
rahman small rna library to the e. histolytica hm-1:imss genome
sequence.
following the same small rna sequence analysis flow-chart as applied to the
e. histolytica hm-1:imss library, the e. histolytica
rahman dataset was analyzed .
overall, there were  <dig>  unique sequence reads, with  <dig> %  of
the sequences found to have been sequenced only once .
small rnas that mapped
to trnas , rrnas  and repetitive elements
were subtracted from the
dataset.
the remaining reads were aligned to the e. histolytica
hm-1:imss genome and to the predicted protein coding genes
.
the mapping of e. histolytica
rahman small rna dataset showed a similar overall distribution pattern as that
of the small rna ehago2- <dig> ip library: many small rna reads from the rahman
library mapped antisense to genes ; the two other main categories of small
rnas were those that mapped sense to genes  and to intergenic regions
.
in addition, the size
distribution of the aligned reads in rahman showed a peak at 27nt
with a 5′-g bias
.
furthermore, there was comparable
sequence depth for the mapped 27nt reads between the hm-1:imss and rahman
libraries .
thus, given that many features are conserved between two
libraries, we concluded that most reads from the rahman size-selected library
may be from ago2- <dig> bound species.
rahman strain.
distribution of small rna
reads cloned from a size-fractionated small rna library from the
non-virulent e. histolytica rahman strain.
small rna
length distribution for reads in the rahman library .
nucleotide frequency at each position for
the aligned 27nt reads in the rahman library reveals a
5′-g predominance.
antisense small rnas
from the e. histolytica rahman strain map to ehstirp1
.
small rna reads from hm-1:imss ehago2- <dig> ip library and
rahman size-fractionated small rna library are shown in red bars  and blue bars ; black arrow represents ehi_ <dig> and its direction.
insert
figures are northern blot analysis using total rna samples from both
strains to show the presence of antisense small rnas in the e.
histolytica rahman strain but not in hm-1:imss strain for
selected ehstirp <dig> probe.
control probe  detected small rna
signal in both samples.
5′-p independent cloning
half of a pyrophosphate sequencing run was performed.
unix tools were
used to remove adaptor and linker sequences.
the bowtie alignment
tool was used to scan for structural rnas, repetitive elements and
for the final genome mapping.
we used e. histolytica
hm-1:imss genome as a reference genome due to the e.
histolytica rahman sequence not being assembled.
the
numbers of reads that map to the genome sequence at each step are
listed.
to be confident that our mapping using the hm-1:imss genome represented the
overall picture for the rahman strain, we also aligned these small rna reads to
the current rahman assembly and compared the number of mapped reads.
aligning
the rahman small rna dataset to the e. histolytica rahman assembly
rather than the hm-1:imss genome only increased the number of mapped reads by 7%
.
overall, this indicates that mapping of small rnas from rahman to
the hm-1:imss genome is representative of the overall picture and the greater
information gained by mapping the small rnas to an annotated assembly outweighed
the negative effects of using a genome sequence from a different strain.
genes with strain-specific expression patterns and roles in virulence have
small rnas that map to them
we cannot directly compare the hm-1:imss and rahman libraries for read
frequencies and patterns of small rna mapping, as they were generated
differently and were sequenced to different depths.
however, common features in
both libraries  suggested that ehago2- <dig> bound species are represented in the rahman
library.
thus, this overlap of small rna coverage between the two libraries
allowed us to look for genes, which might be regulated strain specifically on
the basis of small rna abundance.
an important caveat to the analysis is that
while the presence of small rnas is meaningful in either strain, the absence of
small rnas in the rahman library is less meaningful and could be due to the
limited sequencing depth or use of a size-selected small rna library.
using the same criteria as used for hm-1:imss  the small rna library generated from e. histolytica
rahman identified  <dig> genes with small rnas .
when the genes from these three categories were compared
between the rahman and hm-1:imss strains, we found significant overlap for genes
with antisense and sense/antisense small rnas.
a total of  <dig> genes with only
antisense small rnas were common between the two strains ;  <dig> genes with both antisense and
sense small rnas were common between the two strains .
however, for the genes with sense only
small rnas, no overlap was identified .
these data further support the idea that antisense
small rnas are likely playing roles in conferring strain-specific gene
expression profiles, whereas the small rnas that map sense only to genes are
likely to be random degradation products.
in order to compare gene expression patterns for genes with strain-specific small
rnas, we used previously published microarray data for the two e.
histolytica strains and a two-fold difference in normalized expression
and a p-value < <dig>  as cutoff  <cit> .
of the  <dig> genes that have lower expression in hm-1:imss than in the
rahman strain,  <dig> genes could be potentially regulated by antisense or
antisense/sense small rnas in hm-1:imss .
of the  <dig> genes that have
higher expression in the hm-1:imss strain compared to the rahman strain, 19
genes could be potentially regulated by antisense or antisense/sense small rnas
in rahman .
the e. histolytica serine, threonine, isoleucine, rich protein
was previously identified as a virulence determinant as it has high expression
in e. histolytica virulent strains and no expression in nonvirulent
strains and genetically proven to have a role in virulence  <cit> .
there were  <dig> small rnas that mapped antisense to this gene from
our rahman small rna dataset compared to virtually no antisense small rnas from
the hm-1:imss small rna dataset .
the
distribution of the antisense small rnas to ehstirp gene showed a clear
enrichment at the 5′-end.
we further confirmed the presence of
ehstirp antisense small rnas in the rahman strain  by northern blot analysis  using
total rna samples from two strains.
lastly, we attempted to overexpress a
myc-tagged ehstirp <dig> , which resulted in successful ehstirp
overexpression in the hm-1:imss strain but not in the rahman strain even at very
high levels of drug selection .
the lack of ability to
overexpress the ehstirp gene in the e. histolytica rahman strain that
has abundant antisense small rnas that map to it raises the intriguing
possibility that the antisense small rnas control the silencing of this
virulence gene in the e. histolytica rahman strain.
future studies will
be needed to address this question.
discussion
in this study, we performed pyrosequencing of e. histolytica endogenous
small rnas that associate with the ehago2- <dig> protein in the virulent e.
histolytica hm-1:imss strain and size selected small rnas in the
non-virulent e. histolytica rahman strain.
characterization of these small
rnas showed that they are distinct in size , have preference for
5′-g, and have 5′-polyphosphate termini.
genome mapping revealed that these 27g-rnas are mainly derived from coding genes
with a much smaller population coming from retrotransposons.
comparison of the 27nt
small rna population from e. histolytica strains with varying virulence and
expression profiles demonstrated an inverse correlation between antisense small rna
and gene expression levels, hinting that antisense 27g-small rnas may be involved in
the regulation of strain-specific genes, including known virulence determinants,
such as ehstirp.
we have previously demonstrated that 27nt antisense small rna in e.
histolytica have 5′-polyp termini  <cit> .
in this study, we further characterized small rnas that associate with
ehago2- <dig>  including those small rnas that map both antisense and sense to genes.
for
all the small rnas that were detectable by northern blot analysis, we were able to
show that they have 5′-polyp termini, indicating these abundant
small rna species in e. histolytica are reminiscent of secondary small rna
from c. elegans and ascaris <cit> .
in worms, the biogenesis and stability of
5′-polyphosphate small rnas are dependent on rdrps and wagos
respectively, with these small rnas being a component of the sirna amplification
pathway  <cit> .
signal amplification is controlled by using primary trigger sirnas to
instigate secondary sirnas through rdrp for the enforced silencing, but limiting
secondary sirnas from doing further signal amplification  <cit> .
although small rnas that mapped sense to coding regions were found in
c. elegans and ascaris suum 5'-monop independent libraries,
their existence and functionality were not confirmed; instead they were generally
treated as non-specific degradation products  <cit> .
we have confirmed that in e. histolytica small rnas that map
sense to genes  are detected in northern blot
analyses as discrete band and bear the same 5′-polyp termini.
strand-specific rt-pcrs detected transcripts in both directions for these loci
, implying that rdrp-based
small rna generation could occur for both sense and antisense transcripts in this
parasite.
the e. histolytica genome encodes one full rdrp gene
and two genes with partial rdrp domains .
the functions
of the rdrp genes in e. histolytica rnai pathway are still elusive and need
further investigation.
due to the fact that gene knockout is not feasible in e. histolytica we have
been unable to dissect how the parasite rnai components affect the levels of these
small rnas.
the comparison of 5′-polyp small rnas among the three
organisms in which they have been described  show several differences:  5′-polyp
small rna size in e. histolytica is 27nt, whereas in c. elegans
and ascaris these rnas are 22nt;  the distribution pattern of antisense
small rnas to the targeted gene loci is enriched at the 5′-end for
e. histolytica and ascaris whereas in c. elegans
there is enrichment at the 3′-end of transcripts;  localization
of ehago2- <dig> and bound 27nt small rnas are mostly localized to the parasite nucleus  <cit> , whereas c. elegans 22g-rnas can associate with several
different wagos and have both perinuclear  <cit>  and nuclear localization  <cit> ; and  c. elegans strongly prefers spliced transcripts as rdrp
template for generating 5′-polyp small rnas whereas both mature and
nascent transcripts appear to function as templates in e. histolytica.
future studies aimed at elucidating these different mechanisms are needed.
our previous limited sanger sequencing has shown that small rnas in e.
histolytica largely mapped to the coding genes  <cit> .
our pyrosequencing data further confirmed this mapping, which is in
contrast to other parasitic systems t. gondii, g. intestinalis and t.
brucei where the small rnas are 5′ single phosphate and
mostly derived from repetitive elements, retrotransposons  <cit> .
the genome of e. histolytica contains hundreds of copies of
line and sine elements, with sine elements actively transcribed and line <dig> transcript
detected by northern blot analysis  <cit> .
how retrotransposons are controlled in ameba is not known.
as noted in
our small rna dataset analysis, one way that the rnai pathway could function in this
parasite is to target the unwanted transposon insertion events where segments are
flanked on both ends by inverted repeats  as many small rnas map
to these regions.
whether rnai directly targets retrotransposons in ameba is still
an open question.
further characterization of the other two argonaute proteins
will provide a complete picture of small rna populations in
ameba and their functions in retrotransposon silencing.
the small rna sequencing from different strains  clearly
indicated that expression of a subset of genes, including the virulence factor
ehstirp, appears to be controlled by small rnas in a strain-specific manner.
in
order to determine direct effects of the small rna repertoire on parasite
pathogenesis, the next step will be to perform functional studies to demonstrate
direct roles for these small rnas in regulating strain-specific virulence gene
expression.
CONCLUSIONS
in summary, we present two pyrosequencing small rna datasets from the parasite e.
histolytica: one an ehago2- <dig> ip library from the virulent e.
histolytica hm-1:imss strain and the other a size selected small rna
library from the non-virulent e. histolytica rahman strain.
our analysis
identified a number of new findings:  amebic 27nt small rnas have
5′ g preference;  antisense small rna targeted genes are in
pairs or clustered and notably most of clusters are from segmental duplications d <dig>
d <dig> and d4;  characterization of group ii gene loci shows that both sense and
antisense small rnas have 5′-polyphosphate termini;  small rnas
mapping to introns and exon-exon junctions were found indicating that both spliced
and unspliced mrna can serve as the templates for small rna production;  few
small rnas are found in intergenic regions between paired/clustered genes indicating
that rna transcript from each gene was used as template;  small rnas targeting
retrotransponsons have similar features to the small rna targeting the mrnas, but
are not highly abundant; and  antisense small rnas may contribute to differential
gene expression between virulent and nonvirulent amebic strains including the known
virulence gene ehstirp.
thus, the two small rna datasets in this study will provide
important data for the community to study small rna-mediated gene regulation in this
important human pathogen.

 rna interference is a well-conserved gene regulatory pathway found in most eukaryotes. many important biological functions are controlled by rnai such as developmental regulation, genome protection against viruses and transposons, and dna elimination. small rna molecules, usually – 30nt, are the key elements for rnai. guided by their associated protein complexes, they base-pair to the targeted transcripts or genomic loci to trigger gene silencing at either the transcriptional or post-transcriptional level. in recent years, high throughput sequencing has facilitated the identification of diverse species of small rnas in different organisms. several protozoan parasites such as trypanosoma brucei, toxoplasma gondii, giardia lamblia, trichomonas vaginalis, and entamoeba histolytica contain key genes of the rnai pathway in their genomes. the functions of rnai in parasite biology include retrotransposon control in t. brucei, gene regulation in e. histolytica, and control of antigenic variation in g. lamblia. in t. gondii and t. vaginalis the studies have been limited to endogenous small rna sequencing with no functional studies yet reported. e. histolytica causes dysentery and liver abscesses in humans and affects million people worldwide. the study of this important human parasite has been hampered by lack of standard molecular genetic tools due to the polyploid nature of the e. histolytica genome. recently, several rnai-based gene knockdown approaches -- dsrna/sirna, short-hairpin rna and a transcriptional gene silencing approach in the g parasite strain -- have been established in this organism. we have shown that e. histolytica has a 27nt small rna population, which has 5′-polyphosphate and 3′-oh termini and associates with ehago2-. additionally, we have demonstrated that gene silencing in the e. histolytica g strain is mediated through a sirna pathway. the e. histolytica genome encodes three argonaute proteins of which ehago2- is highly expressed and associates with 27nt small rnas. in this report, we immunoprecipitated small rnas bound to ehago2- and sequenced them using a high throughput pyrosequencing approach, which generated over small rna reads. analysis of these endogenous small rnas revealed that their peak-length is 27nt and that there was a strong g bias at the 5′-nucleotide. genome analysis showed that small rnas largely mapped to annotated protein coding genes, which can be categorized into three groups: genes with only antisense small rnas; genes with both antisense and sense small rnas; and genes with only sense small rnas. biochemical analysis revealed that both sense and antisense small rnas have 5′-polyp termini. strand specific rt-pcr showed both sense and antisense transcripts can be detected for group ii gene loci, suggesting that bi-directional transcripts are available to serve as templates for small rna generation. identification of small rnas that map to introns as well as exon-exon junctions indicated that e. histolytica rna-dependent rna polymerase could use both nascent and mature transcript as a template for generating small rnas. whole genome microarray data indicated that most protein coding genes with large numbers of antisense small rnas are not expressed, indicating that antisense small rnas likely play a role in gene silencing. we further sequenced a size-fractionated small rna library from the non-virulent e. histolytica rahman strain and identified strain-specific patterns of antisense small rnas mapping to a virulence gene in a manner consistent with its regulation by small rnas. in summary, we have characterized the endogenous small rna populations from a virulent and non-virulent strain of e. histolytica using a high-throughput small rna pyrosequencing approach. our analysis indicates that the small rna pathway likely regulates expression of strain-specific genes in this parasite system, including some virulence determinants.  high-throughput pyrophosphate sequencing of ehago2-2-bound small rnas a library of ehago2- associated small rnas was generated from rna obtained by immunoprecipitation with anti-myc antibody in e. histolytica hm-1:imss cells stably expressing myc-tagged ehago2- we have previously shown that ehago2- specifically associates with 27nt small rnas, which have 5′-polyp and 3′-oh termini. thus, the small rna library was generated using a 5′-p independent cloning approach. genome sequencer process-specific a and b adaptors were incorporated into the cdna small rna library by pcr and a full sequencing run was performed generating a total of sequences. using the small rna sequence analysis workflow, we removed adaptor and linker sequences and limited the size to 15-40nt. the resulting output contained sequences that represented unique sequence reads. about % of the unique sequences were cloned only once indicating that the current sequencing was far from saturating. library from e. histolytica hm-1:imss 5′-p independent cloning one pyrophosphate sequencing run was performed. unix tools were used to remove adaptor and linker sequences. the bowtie alignment tool was used to scan for structural rnas, repetitive elements and for the final genome mapping. the numbers of reads that map to the genome sequence at each step are listed. we used the bowtie alignment tool to filter out reads that mapped to structural rnas: trna, rrna and repetitive elements. the remaining dataset was mapped to the e. histolytica hm-1:imss genomic sequence. a total of reads mapped to the genome with reads mapping to open reading frames and reads mapping to a single gene locus. this dataset targets ~4% of annotated protein coding genes in the e. histolytica genome. the genome mapping revealed some striking features. first, many small rna reads mapped antisense to annotated protein coding genes; small rnas that mapped to intergenic regions and sense to protein coding genes were less common. second, small rnas mapping to sine/line retrotransposon elements and other potential repetitive regions in the genome only accounted for 5% and % of the reads respectively. this demonstrates that ehago2- is not primarily associated with small rnas derived from transposons, in contrast to the single t. brucei argonaute protein, which has been shown to associate with small rnas that map primarily to transposons and are thought to control their expression. third, the mapping of small rna to the genome indicated that small rna reads tend to be derived from a small number of genomic locations or “hot spots”. when the genome was scanned using a 500 bp window, the majority of small rnas arose from % of total windows in genome. this suggests that the small rnas could associate with certain genomic features such as repeat regions, centromeric or telomeric regions, which have been shown to be a source of small rnas in other systems. in summary, our pyrosequencing data indicated that ehago2-2 associates with an abundant endogenous small rna population, which is largely derived from the predicted protein coding genes in e. histolytica. e. histolytica hm-1:imss trophozoites. the composition of small rnas from the ehago2- ip small rna library that match the e. histolytica genome annotation in the hm-1:imss strain. small rna length distribution for all reads and all reads remaining after filtering out those that originate from structural rnas and repetitive elements reads is shown. nucleotide frequency at each position for the aligned 27nt reads reveals a 5′-g predominance small rnas are largely 27nt size with a striking 5′-g bias we analyzed the size distribution and nucleotide composition of small rnas and found that all reads peaked sharply at 27nt, consistent with the size of small rnas previously noted to associate with ehago2-. when nucleotide frequency was plotted at each position for the 27nt population we noticed a striking 5′-g bias. the nucleotide frequency for the 26nt and 28nt small rnas also shows a 5′-g propensity, but this was not the case for 17nt small rnas indicating that the smaller sequences are likely degradation products. given that the e. histolytica genome is very at-rich, the 5′-g bias in small rnas is remarkable when compared to all the remaining plotted positions for small rnas. a 5′-g bias in small rnas has been reported in two other organisms, i.e. c. elegans and ascaris, where 22g populations with 5′-polyp termini are defined as secondary sirnas and thought to be generated by rdrp. thus, we reason that the 5′-g biased 27nt small rnas are likely rdrp-related and function as silencing sirnas in e. histolytica. small rna distribution patterns in the genome the e. histolytica genome was first published in and a second version including new assemblies and reannotation was released in. the current genome assembly is still in scaffold stage, which contains supercontigs and genes. to gain an overview of small rna distribution in the genome, we mapped small rnas using the bowtie alignment tool. we identified supercontigs that had ≥ small rnas, and which accounted for 52% of the genome size and which contained 96% of the total small rnas. specifically, the small rnas were highly enriched on supercontigs that although only  mb in total size contained ~50% of all sequenced small rnas. as the structure of the genome is unknown at present, we do not know if the supercontigs that were enriched in small rnas belong to centromeric or telomeric regions. to analyze the overall small rna density distribution on the genome, we scanned the genome using a 500 bp window, and counted numbers of small rnas in each window. we defined a “hot spot” as containing ≥ small rnas per window; using these parameters, there were “hot spot” windows out of genomic windows. the graphic views of “hot spot” distribution further revealed that most small rnas arose either from some large clusters or from isolated peaks. there are regions of the genome that had only a few mapped small rnas. we analyzed the protein coding genes to which small rnas mapped, and found that many genes had only a few small rnas that mapped to them and hence are likely artifacts. thus, we tested our dataset with different cutoffs for the number of small rnas mapping to a gene; we used four measures. for each cutoff, we identified the number of protein coding genes in each category and plotted the microarray expression value for these genes. for the two least stringent criteria, we observed no significant difference in the microarray expression value for the three categories of protein coding genes. however, when we used either the ≥ or ≥ small rna cutoff, we identified significantly lower expression among genes with antisense or sense/antisense small rnas compared to genes with sense small rnas. the total number of protein coding genes using either cutoff was relatively similar. to be as stringent as possible, we decided to use a cutoff of ≥ small rnas mapping to a gene for further analysis. overall, protein coding genes had ≥ small rnas that mapped to them. these protein coding genes could be categorized into three groups: genes with only antisense small rnas; genes with both antisense and sense small rnas; and genes with only sense small rnas. most genes in group i and ii are annotated as hypothetical proteins. however, a few gene families were represented including aig family proteins, beta-amylase, deoxyuridine 5′-triphosphate nucleotidohydrolase domain proteins, dna polymerase, and c2 domain proteins. in order to determine whether protein coding genes with small rnas are in proximity to each other, we characterized the patterns of genes to which small rnas map. a cluster is defined as ≥ contiguous genes. a pair is defined as contiguous genes that are ≤1000 bp apart. there are a total of protein coding genes that have ≥ small rnas and of these the majority are in clusters or in pairs. these clustered/paired genes were largely in group i and ii categories. we next looked at transcript orientation in the paired genes. the protein coding genes in pairs had divergent pairs, convergent pairs, and tandem pairs. a total of protein coding genes are unpaired with 63 genes in group i, genes in group ii, and genes in group iii. when we characterized clustered protein coding genes with small rnas, we identified clusters, which ranged in size from a cluster of 3 genes to clusters as large as genes. a substantial number of clusters are in previously identified regions of d d and d genome duplication, which are segmental duplications with each segment flanked on both ends by inverted repeats such as ir/ehere1/ehlines. clusters that are not in regions of d1-d genome duplications are still flanked by repetitive elements at either one or both ends. thus, clustered genes are more likely to be associated with repetitive elements. in order to determine whether small rnas are enriched on paired or clustered protein coding genes or in the intergenic dna regions, we calculated the small rna density on these paired/clustered genes as well as the intergenic regions between genes. this was calculated as small rna/bp. we identified that the density of small rnas mapping to intergenic regions was significantly lower compared to small rna density mapping to paired/clustered genes. this indicates that small rna synthesis is most likely templated using a given gene rather than a long template covering several genes. for intergenic regions that had high small rna density, we found these small rnas are often in discrete sections or adjacent to predicted genes. thus, we postulate that this may be due to small rnas mapping to an unannotated gene or utrs. in summary, our analysis suggests that the small rna targeted protein coding genes tend to be in pairs or clusters, and that clusters of genes with small rnas are more often associated with repetitive elements. small rna density on paired/clustered genes versus intergenic regions implies that it is unlikely for either dna or a long transcript covering several genes to be used as a template, but rather that transcript derived from each gene is the most likely template. small rna distribution patterns within protein coding genes we have previously shown that small rnas that map antisense to protein coding genes tend to be most abundant toward the 5′-end of genes. however, that analysis was done using a very limited dataset of small rnas generated from sanger sequencing. our new pyrophosphate sequencing dataset enabled us to examine this observation on a larger scale. using the stringent criteria of ≥ small rnas mapping to a gene, a total of 226 protein coding genes were categorized as group i. we plotted small rna distribution along each gene. there was a clear trend showing that most antisense small rnas mapped toward the 5′-termini of predicted genes. this trend holds true for most targeted protein coding genes and was not caused by a few genes with a high number of small rnas at the 5′-end. for the 5′-polyp small rnas in ascaris and c. elegans, there is a clear difference of small rna distribution on their corresponding mrna targets. in c. elegans 22g-rnas are mostly enriched at 3′-end of the mrna, while ascaris 22g-rnas are distributed toward the 5′-end of mrnas. our results indicate that the small rna distribution pattern for the antisense small rnas in e. histolytica is more similar to that in ascaris as compared to c. elegans. the 5′-bias of antisense small rnas could reflect the heavy recruitment of rdrp complexes to these regions, and the exact mechanism of rdrp in generating secondary antisense small rnas is largely unknown at present. protein coding genes. each small rna read was assigned a position value based on the position of the starting nucleotide along the gene. the gene length for each protein coding gene was normalized to one. total number of small rna reads was plotted from 5′ to 3′ according to their relative position within all genes. small rna distribution on genes with only antisense small rnas. small rna distribution on genes with both antisense and sense small rnas. small rna distribution on genes with only sense small rnas. grey bars: antisense small rnas; white bars: sense small rnas. for the group ii genes, we similarly plotted the small rna distribution along each gene. we noted that antisense small rnas were distributed with a 5′-enrichment, while the sense small rna distribution pattern was more heterogeneous. additionally, we noted that for group ii genes, in most cases the number of antisense small rnas was greater than the number of sense small rnas for each gene locus. genes with both antisense and sense small rnas. ehi_ is depicted with the small rnas that map to it; each bar represents one unique small rna. the positions of selected probes used for small rna northern blot analysis are represented by bars and numbers. the positions of the f and r primers, used to generate cdna for the strand-specific rt-pcr, are shown. northern blot analysis detected signal for antisense small rnas and sense small rnas. sense small rnas have 5′-polyp termini. 10 μg small rna enriched sample was treated with terminator or capping enzyme. probes were used for northern blot analysis. a control oligonucleotide that is labeled with a 5′-monop is degraded by terminator and has no change in size with capping enzyme, as expected. strand specific rt-pcr demonstrates both sense and antisense transcripts for ehi_ cdna was generated using f and r primer as well as oligo dt primer. rt-pcr reveals both antisense and sense transcripts with antisense transcript at lower abundance than sense transcript. both rt and control reactions lacking rt are shown. in contrast, when small rna distribution was plotted for group iii genes, it became apparent that sense small rnas were enriched towards the 3′-end of each gene. we noticed that genes with only sense small rnas mainly code for four highly expressed gene families; gal/galnac lectin; three protein kinase families; and hypothetical proteins ). since our cloning method will capture all types of small rnas, degradation products with 5′-oh species would also be cloned; this may suggest that at least some of the sense small rnas might be mrna degradation products. however, we found no sense small rnas to other highly expressed genes, indicating some specificity for sense small rnas to these specific loci. since the mapping of sense small rnas to many genes often had abrupt boundaries within/near 3′ end of genes and sometimes extended into intergenic regions, we felt that some mapping artifacts could be due to poor genome quality in these regions. as the e. histolytica genome is not complete at present, we cannot rule out whether or not these sense small rnas are derived from some other loci. although sense small rnas have been identified in almost all cloning libraries, these types of small rnas have largely been ignored since it is hard to evaluate their identity as true endogenous small rnas versus non-specific hydrolysis fragments. in both c. elegans and ascaris suum, where 5′-polyp small rnas were found, sense small rnas were identified but were not characterized. for the e. histolytica group iii genes that have only sense small rnas mapped to them we have not made any assumptions and have not further characterized the structure, derivation, or function of this category of sense small rnas. antisense and sense small rnas have 5′-polyphosphate termini the secondary 5′-polyp small rnas in nematodes are thought to be generated by an amplified gene silencing mechanism, likely through rdrp. we have previously demonstrated that in e. histolytica both endogenous and exogenously triggered antisense small rnas have 5′-polyp termini. since our analysis to date had focused on the antisense small rnas, we wanted to determine whether the small rnas that map sense to genes also have 5′-polyp termini. for this purpose, we analyzed small rnas that mapped sense to the ehi_ locus. this gene is highly enriched for both antisense and sense small rnas with small rna coverage extending into the apparent 5′-upstream region. northern blot analysis detected signal at 27nt with probes that detect antisense small rnas and with probes that detect sense small rnas. all the blots were performed using the same membrane, and therefore the relative band intensity reflects the abundance of small rnas. we observed a correlation of 5'-enriched distribution of small rnas with the intensity of probe 1 > probe 2 > probe we then performed a terminator exonuclease assay and a 5′-end capping assay on the total rna sample. the signal for sense small rnas were resistant to terminator treatment and shifted up after capping assay, indicating that these small rnas that map sense to the ehi_ locus have 5′-polyp termini. probe which would detect antisense small rnas had the expected biochemical features consistent with 5′-polyp termini. the control was, as expected, degraded by terminator enzyme and unaffected by treatment with capping enzyme. we further examined a second example: the locus that contains both antisense and sense small rnas to ehi_ and ehi_ as well as one potential unannotated gene. probes for detecting antisense and sense small rnas were chosen for ehi_ northern blot analysis showed signal for both probes at ~27nt; the sense small rna was resistant to terminator exonuclease and thus has 5′-polyphosphate termini. thus, for the two loci tested we determined that the sense small rnas also have 5′-polyp termini. the biochemical features of sense small rnas having 5′-polyp termini was unexpected. analysis for pairing between antisense and sense small rnas showed no enriched pairs for these gene loci. in a typical rnai pathway, dsrna is chopped into a sirna duplex, where enriched pairing between antisense and sense small rnas can be found. the lack of pairing between antisense and sense small rnas in group ii genes and the 5′-polyp termini for both antisense and sense small rnas indicates that these small rnas could be individually processed from bidirectional transcripts at these loci. to determine if this may have occurred, we performed strand-specific rt-pcr for ehi_ ehi_ and ehi_ at all three loci, both sense and antisense transcripts can be detected, albeit with antisense transcripts at much lower level than the sense transcript. overall transcript levels as assayed by rt-pcr correlated with the abundance of antisense and sense small rnas mapped to these loci. we further applied the strand-specific rt-pcr assay to two additional protein coding genes, which had both antisense and sense small rnas and identified transcripts in both directions for these loci. thus, we conclude that although bidirectional transcription has not been previously documented in e. histolytica, both antisense and sense small rnas are likely templated from bidirectional transcripts and generated by an rdrp-dependent mechanism resulting in small rnas with 5′-polyphosphate termini. natural antisense transcripts have been shown to be a major source of sirna generation in drosophila melanogaster and in plants. in the parasite, giardia lamblia, bidirectional transcription produces abundant sterile antisense transcripts. further studies to characterize the extent of natural antisense transcripts in e. histolytica and the mechanism for generation of 5′-polyp small rnas are needed. our demonstration that small rnas to group ii genes have 5′-polyp termini and strand-specific rt-pcr detecting transcripts in both directions indicates that these sense small rnas are likely generated from antisense transcript, and indicates that some portion of “sense” small rnas in our dataset although called “sense”, are truly antisense to the transcript derived from the opposite strand of the examined gene. small rnas are derived from both unspliced and spliced transcripts in order to identify whether small rnas map to spliced or unspliced transcripts, we downloaded both genomic and mrna sequences for all e. histolytica protein coding genes with at least one predicted intron. small rnas mapping to introns are defined as those that map to the genomic gene sequence but not the mrna sequence. small rnas mapping to exon-exon junctions are those that map to mrna sequence but not the genomic gene sequence. we found a total of small rna reads that spanned exon-exon junctions and small rna reads that mapped to predicted introns. we further examined the protein coding genes with at least small rnas and which also have at least one intron. for this list, we first checked for potential “false introns” caused by genome sequence error, and excluded ehi_ ehi_ and ehi_ as they have many ns in their intron sequences. we then checked the predicted intronic sequence in the remaining genes for an in-frame stop codon or frame disruption, as this strongly suggests that the intron is correctly predicted. lastly, we checked for paralogs within these genes. with these criteria, we examined the three categories of genes to which small rnas mapped to identify small rnas that mapped to exons, introns and exon-exon junctions. for the group i genes, unique genes had small rnas that mapped to predicted introns and among them, unique genes also had small rnas that mapped to exon-exon junctions. for the group ii genes, unique genes had antisense small rnas that mapped to introns and of these also had sense small rnas that mapped to introns. none of the genes in this category had small rnas that mapped to exon-exon junctions. for the group iii genes, unique genes had small rnas mapped to predicted introns, but none mapped to exon-exon junctions. overall, we made a number of observations: a greater number of small rnas mapped to exons than to the introns for all three groups of genes; all but one intron-containing gene in groups i and ii had small rnas that mapped to introns; and only a limited number of genes had small rnas that mapped to exon-exon junctions. these data indicate that both spliced and unspliced transcripts are capable of being used as templates to produce small rnas in e. histolytica. as an example, the mapping of small rnas to exons, introns and exon-exon junctions are shown for ehi_ and ehi_197360 genes. further calculations of the small rna density revealed four-fold greater density of small rnas in exons than in introns [ehi_ exon vs. intron; ehi_ exon vs. intron ]. the difference could suggest that spliced transcripts are preferred as templates to unspliced transcripts, or alternatively may simply be a reflection of the ratio of spliced and unspliced transcripts available in the cell. in c. elegans, ego- an rdrp, is critical for c. elegans germline development and is responsible for producing 5′-polyp antisense small rnas from mrna-derived loci. small rna sequencing has shown that small rnas often span exon-exon junctions and rarely map to introns, indicating ego- uses processed mrna as a template. rdrp could theoretically template on genomic dna, nascent transcripts, or processed mrnas. small rnas that map to exon-exon junctions provide evidence that a spliced mrna template is used to generate these small rnas, whereas small rnas that map to introns indicate that non-spliced templates can also be used to generate small rnas. based on the observations that there are small rna free genomic regions between genes with antisense small rnas, and that many more small rnas map to exons than introns, we conclude that the e. histolytica rnai machinery prefers mature transcript as a template for generating small rnas. however, the machinery in e. histolytica also seems capable of using unspliced transcripts as template, although at reduced levels. whether this is indicative of the inherent preference of the e. histolytica machinery or due instead to the low abundance of unspliced mrna is not clear at present. small rnas that map to trnas, rrnas and retrotransposon elements in order to identify small rnas that map to the trnas, rrnas and retrotransposon elements, we followed the outline in additional file 1: figure s e. histolytica has uniquely organized trna genes that are in multiple tandem-array units, likely arranged at subtelomeric regions and spaced by tandem repeats of at-rich sequences. the e. histolytica rrna genes reside on an extrachromosomal circular plasmid and two rrna transcription units are organized as inverted repeats. we mapped the small rna reads to the trna repeat units and the rrna plasmid. we found that nearly all small rna reads were in the sense orientation to the coded trnas and rrnas. additionally, we plotted the size distribution and nucleotide frequency for rrna reads and noted that small rnas in these categories did not peak at 27nt, and did not have a 5′-g enrichment indicating that these small rnas are most likely degradation products resulting from these highly expressed structural rnas. however, we are aware of some recent reports showing that small rnas could originate from trnas and snornas. upon a closer examination of the trna reads, we noticed a slight peak at 27nt with some degree of 5′-g enrichment for this 27nt population only. whether or not this indicates that these are functional small rnas in entamoeba needs further study. transposons and repetitive dna are abundant in entamoeba and hundreds of copies of the long interspersed nuclear elements and short interspersed nuclear elements can be found in the e. histolytica genome. our small rna dataset contains 5% of reads that mapped to line and sine elements. analysis of the lengths of these small rnas showed two peaks. when nucleotide composition is plotted, the 5′-g propensity is apparent for the 27nt peak, but not for the 17nt peak. thus, the 27nt small rna population that maps to line/sine elements had features similar to those that map to coding regions and are likely not artifacts. as an example, we mapped small rnas to the ehrle sequence, which has been categorized in the ehline1 family. the small rnas are scattered along the whole region on both strands and cover the whole ehrle unit, with a slight increase in small rnas near each end. we got a positive signal from northern blot analysis using several probes to retrotransposon elements although the size by northern blot analysis was slightly higher than the cloned small rna. this indicates that small rnas could derive from these retrotransposon elements. the mapping of small rnas to d1-d repetitive segments showed a large number of small rna reads on d d and d segments but not on d segments. annotated protein coding genes in these duplication regions appear to be covered by large numbers of antisense small rnas, forming a large cluster. thus, although the overall numbers of small rnas that associate with ehago2- and map to repeat or retrotransposon elements is low, they may play a functional role in controlling genome stability as has been shown in other systems. alternatively, small rnas may have a role in controlling these retrotransposon elements but may do so by associating with the two other argonaute proteins in e. histolytica. a global assessment of genes potentially regulated by small rnas in e. histolytica we have previously shown an inverse correlation between gene expression and antisense small rna abundance raising the intriguing possibility that antisense small rnas may mediate target gene silencing in e. histolytica. however, those data were on a very limited scale due to the very limited set of sequenced small rnas. the pyrosequencing dataset allowed us to assess the potential genome-wide affects of these small rnas by comparing small rna abundance with microarray expression data. we used previously published microarray data from e. histolytica hm-1:imss trophozoites. the analysis was conducted for the three groups of protein coding genes with distinct small rna mapping patterns. for group i genes, there were protein coding genes that met the criteria; of these, genes are represented on the microarray. we plotted the number of mapped small rna reads for each gene as a function of normalized microarray expression data and identified that most genes in this category are not expressed. for group ii genes there are genes that met our criteria and are represented on the microarray and most of these genes are also not expressed. in order to determine whether these genes are expressed under other conditions, we compared the expression profiles of these genes across all other conditions tested. of these genes, were expressed in a strain-specific manner, were regulated under various culture conditions, and were regulated during stage conversion. overall, a total of protein coding genes are not expressed in e. histolytica hm-1:imss trophozoites under standard in vitro growth conditions. based on our sequencing data we estimated that small rnas target about 41% of these protein coding genes. of these 112 genes, a large proportion change in expression profile under one or more conditions examined indicating that they are not permanently silenced and may be regulated by small rnas. of small rnas targeting to a gene. genes are plotted using the number of mapped small rnas and their normalized microarray expression levels. the dashed line indicates the cutoff for gene expression generally detectable by rt-pcr. plot for group i genes. plot for group ii genes. plot for group iii genes. box-and-whisker expression plots for genes in groups i, ii, iii as well as all genes represented on the microarray. the top and bottom ends of each box represent the 75th and 25th percentile, respectively; the middle line represents the median value. for the genes in group iii, genes met our criteria with represented on the microarray. plotting the number of mapped reads for each gene as a function of its normalized microarray gene expression value showed no direct link between numbers of sense small rnas to a gene and the expression level of that gene. we reasoned that for genes with high expression value, the sense small rnas may represent degradation products. however, for genes with very low mrna expression, we have no good explanation on how these sense small rnas were generated. whether this represents an artifact of errors in genome assembly or some other factor is not clear at present. interestingly, none of these genes are regulated under other conditions tested. in summary, the strongest correlation between gene expression and small rna abundance was for genes that had either abundant antisense small rnas or abundant sense and antisense small rnas. although simply correlative at present, the inverse correlation between antisense small rna abundance and gene expression suggests that antisense small rnas mediate target gene silencing in e. histolytica and are potentially involved in gene regulation under various conditions. small rna sequencing from the nonvirulent e. histolytica rahman strain it has been observed that pathogenicity varies greatly among different e. histolytica strains. e. histolytica hm-1:imss is a virulent strain, while e. histolytica rahman is a nonvirulent strain. we have previously demonstrated that gene expression profiles are substantially different between these two strains and have used the distinct strain-specific expression profiles to identify virulence genes. given the data above, and to explore whether small rnas play a role in strain-specific and/or virulence gene regulation, we constructed a small rna library from trophozoites of the e. histolytica rahman strain. we visualized the small rna populations in e. histolytica rahman by separating total rna on a 12% denaturing polyacrylamide gel followed by sybr gold staining and visualized an abundant 27nt small rna population. a small rna library was constructed from size-fractionated rna using a 5′-p independent cloning approach and limited pyrophosphate sequencing was performed generating reads. for the purpose of mapping, we used e. histolytica hm-1:imss genome as a reference genome rather than the current e. histolytica rahman assembly, based on the following facts: the current e. histolytica rahman genome assembly is in a preliminary stage containing small contigs and is unannotated, there is a high similarity between these two strains and one previous study has estimated that only out of a sample of genes were identified as highly or significantly divergent, and affymetrix platform microarrays found no difference in overall hybridization efficiency levels compared to hm-1:imss, indicating a high level of sequence identity for the protein coding genes. we realize that sequence differences between the e. histolytica hm-1:imss and rahman strains may cause us to lose some data. however, the advantages of being able to map to an annotated genome and thus determine how many small rnas map to protein coding genes and to intergenic regions were significant enough that we proceeded with the data generated by aligning the e. histolytica rahman small rna library to the e. histolytica hm-1:imss genome sequence. following the same small rna sequence analysis flow-chart as applied to the e. histolytica hm-1:imss library, the e. histolytica rahman dataset was analyzed. overall, there were unique sequence reads, with % of the sequences found to have been sequenced only once. small rnas that mapped to trnas, rrnas and repetitive elements were subtracted from the dataset. the remaining reads were aligned to the e. histolytica hm-1:imss genome and to the predicted protein coding genes. the mapping of e. histolytica rahman small rna dataset showed a similar overall distribution pattern as that of the small rna ehago2- ip library: many small rna reads from the rahman library mapped antisense to genes; the two other main categories of small rnas were those that mapped sense to genes and to intergenic regions. in addition, the size distribution of the aligned reads in rahman showed a peak at 27nt with a 5′-g bias. furthermore, there was comparable sequence depth for the mapped 27nt reads between the hm-1:imss and rahman libraries. thus, given that many features are conserved between two libraries, we concluded that most reads from the rahman size-selected library may be from ago2- bound species. rahman strain. distribution of small rna reads cloned from a size-fractionated small rna library from the non-virulent e. histolytica rahman strain. small rna length distribution for reads in the rahman library. nucleotide frequency at each position for the aligned 27nt reads in the rahman library reveals a 5′-g predominance. antisense small rnas from the e. histolytica rahman strain map to ehstirp1. small rna reads from hm-1:imss ehago2- ip library and rahman size-fractionated small rna library are shown in red bars and blue bars; black arrow represents ehi_ and its direction. insert figures are northern blot analysis using total rna samples from both strains to show the presence of antisense small rnas in the e. histolytica rahman strain but not in hm-1:imss strain for selected ehstirp probe. control probe detected small rna signal in both samples. 5′-p independent cloning half of a pyrophosphate sequencing run was performed. unix tools were used to remove adaptor and linker sequences. the bowtie alignment tool was used to scan for structural rnas, repetitive elements and for the final genome mapping. we used e. histolytica hm-1:imss genome as a reference genome due to the e. histolytica rahman sequence not being assembled. the numbers of reads that map to the genome sequence at each step are listed. to be confident that our mapping using the hm-1:imss genome represented the overall picture for the rahman strain, we also aligned these small rna reads to the current rahman assembly and compared the number of mapped reads. aligning the rahman small rna dataset to the e. histolytica rahman assembly rather than the hm-1:imss genome only increased the number of mapped reads by 7%. overall, this indicates that mapping of small rnas from rahman to the hm-1:imss genome is representative of the overall picture and the greater information gained by mapping the small rnas to an annotated assembly outweighed the negative effects of using a genome sequence from a different strain. genes with strain-specific expression patterns and roles in virulence have small rnas that map to them we cannot directly compare the hm-1:imss and rahman libraries for read frequencies and patterns of small rna mapping, as they were generated differently and were sequenced to different depths. however, common features in both libraries suggested that ehago2- bound species are represented in the rahman library. thus, this overlap of small rna coverage between the two libraries allowed us to look for genes, which might be regulated strain specifically on the basis of small rna abundance. an important caveat to the analysis is that while the presence of small rnas is meaningful in either strain, the absence of small rnas in the rahman library is less meaningful and could be due to the limited sequencing depth or use of a size-selected small rna library. using the same criteria as used for hm-1:imss the small rna library generated from e. histolytica rahman identified genes with small rnas. when the genes from these three categories were compared between the rahman and hm-1:imss strains, we found significant overlap for genes with antisense and sense/antisense small rnas. a total of genes with only antisense small rnas were common between the two strains; genes with both antisense and sense small rnas were common between the two strains. however, for the genes with sense only small rnas, no overlap was identified. these data further support the idea that antisense small rnas are likely playing roles in conferring strain-specific gene expression profiles, whereas the small rnas that map sense only to genes are likely to be random degradation products. in order to compare gene expression patterns for genes with strain-specific small rnas, we used previously published microarray data for the two e. histolytica strains and a two-fold difference in normalized expression and a p-value < as cutoff. of the genes that have lower expression in hm-1:imss than in the rahman strain, genes could be potentially regulated by antisense or antisense/sense small rnas in hm-1:imss. of the genes that have higher expression in the hm-1:imss strain compared to the rahman strain, 19 genes could be potentially regulated by antisense or antisense/sense small rnas in rahman. the e. histolytica serine, threonine, isoleucine, rich protein was previously identified as a virulence determinant as it has high expression in e. histolytica virulent strains and no expression in nonvirulent strains and genetically proven to have a role in virulence. there were small rnas that mapped antisense to this gene from our rahman small rna dataset compared to virtually no antisense small rnas from the hm-1:imss small rna dataset. the distribution of the antisense small rnas to ehstirp gene showed a clear enrichment at the 5′-end. we further confirmed the presence of ehstirp antisense small rnas in the rahman strain by northern blot analysis using total rna samples from two strains. lastly, we attempted to overexpress a myc-tagged ehstirp, which resulted in successful ehstirp overexpression in the hm-1:imss strain but not in the rahman strain even at very high levels of drug selection. the lack of ability to overexpress the ehstirp gene in the e. histolytica rahman strain that has abundant antisense small rnas that map to it raises the intriguing possibility that the antisense small rnas control the silencing of this virulence gene in the e. histolytica rahman strain. future studies will be needed to address this question. discussion in this study, we performed pyrosequencing of e. histolytica endogenous small rnas that associate with the ehago2- protein in the virulent e. histolytica hm-1:imss strain and size selected small rnas in the non-virulent e. histolytica rahman strain. characterization of these small rnas showed that they are distinct in size, have preference for 5′-g, and have 5′-polyphosphate termini. genome mapping revealed that these 27g-rnas are mainly derived from coding genes with a much smaller population coming from retrotransposons. comparison of the 27nt small rna population from e. histolytica strains with varying virulence and expression profiles demonstrated an inverse correlation between antisense small rna and gene expression levels, hinting that antisense 27g-small rnas may be involved in the regulation of strain-specific genes, including known virulence determinants, such as ehstirp. we have previously demonstrated that 27nt antisense small rna in e. histolytica have 5′-polyp termini. in this study, we further characterized small rnas that associate with ehago2- including those small rnas that map both antisense and sense to genes. for all the small rnas that were detectable by northern blot analysis, we were able to show that they have 5′-polyp termini, indicating these abundant small rna species in e. histolytica are reminiscent of secondary small rna from c. elegans and ascaris. in worms, the biogenesis and stability of 5′-polyphosphate small rnas are dependent on rdrps and wagos respectively, with these small rnas being a component of the sirna amplification pathway. signal amplification is controlled by using primary trigger sirnas to instigate secondary sirnas through rdrp for the enforced silencing, but limiting secondary sirnas from doing further signal amplification. although small rnas that mapped sense to coding regions were found in c. elegans and ascaris suum 5'-monop independent libraries, their existence and functionality were not confirmed; instead they were generally treated as non-specific degradation products. we have confirmed that in e. histolytica small rnas that map sense to genes are detected in northern blot analyses as discrete band and bear the same 5′-polyp termini. strand-specific rt-pcrs detected transcripts in both directions for these loci, implying that rdrp-based small rna generation could occur for both sense and antisense transcripts in this parasite. the e. histolytica genome encodes one full rdrp gene and two genes with partial rdrp domains. the functions of the rdrp genes in e. histolytica rnai pathway are still elusive and need further investigation. due to the fact that gene knockout is not feasible in e. histolytica we have been unable to dissect how the parasite rnai components affect the levels of these small rnas. the comparison of 5′-polyp small rnas among the three organisms in which they have been described show several differences: 5′-polyp small rna size in e. histolytica is 27nt, whereas in c. elegans and ascaris these rnas are 22nt; the distribution pattern of antisense small rnas to the targeted gene loci is enriched at the 5′-end for e. histolytica and ascaris whereas in c. elegans there is enrichment at the 3′-end of transcripts; localization of ehago2- and bound 27nt small rnas are mostly localized to the parasite nucleus, whereas c. elegans 22g-rnas can associate with several different wagos and have both perinuclear and nuclear localization; and c. elegans strongly prefers spliced transcripts as rdrp template for generating 5′-polyp small rnas whereas both mature and nascent transcripts appear to function as templates in e. histolytica. future studies aimed at elucidating these different mechanisms are needed. our previous limited sanger sequencing has shown that small rnas in e. histolytica largely mapped to the coding genes. our pyrosequencing data further confirmed this mapping, which is in contrast to other parasitic systems t. gondii, g. intestinalis and t. brucei where the small rnas are 5′ single phosphate and mostly derived from repetitive elements, retrotransposons. the genome of e. histolytica contains hundreds of copies of line and sine elements, with sine elements actively transcribed and line transcript detected by northern blot analysis. how retrotransposons are controlled in ameba is not known. as noted in our small rna dataset analysis, one way that the rnai pathway could function in this parasite is to target the unwanted transposon insertion events where segments are flanked on both ends by inverted repeats as many small rnas map to these regions. whether rnai directly targets retrotransposons in ameba is still an open question. further characterization of the other two argonaute proteins will provide a complete picture of small rna populations in ameba and their functions in retrotransposon silencing. the small rna sequencing from different strains clearly indicated that expression of a subset of genes, including the virulence factor ehstirp, appears to be controlled by small rnas in a strain-specific manner. in order to determine direct effects of the small rna repertoire on parasite pathogenesis, the next step will be to perform functional studies to demonstrate direct roles for these small rnas in regulating strain-specific virulence gene expression.  in summary, we present two pyrosequencing small rna datasets from the parasite e. histolytica: one an ehago2- ip library from the virulent e. histolytica hm-1:imss strain and the other a size selected small rna library from the non-virulent e. histolytica rahman strain. our analysis identified a number of new findings: amebic 27nt small rnas have 5′ g preference; antisense small rna targeted genes are in pairs or clustered and notably most of clusters are from segmental duplications d d and d4; characterization of group ii gene loci shows that both sense and antisense small rnas have 5′-polyphosphate termini; small rnas mapping to introns and exon-exon junctions were found indicating that both spliced and unspliced mrna can serve as the templates for small rna production; few small rnas are found in intergenic regions between paired/clustered genes indicating that rna transcript from each gene was used as template; small rnas targeting retrotransponsons have similar features to the small rna targeting the mrnas, but are not highly abundant; and antisense small rnas may contribute to differential gene expression between virulent and nonvirulent amebic strains including the known virulence gene ehstirp. thus, the two small rna datasets in this study will provide important data for the community to study small rna-mediated gene regulation in this important human pathogen. 
exists, however, the depth and diversity of the small rna population remains to characterize the small rna population that associates with e. small rna populations may regulate virulence genes. exon-exon junctions, and small rna targeted genes that are clustered small rna mediated gene silencing is a well-conserved regulatory pathway. analysis revealed new features of the 27nt small rnas including the 5′-g predominance, distinct small rna distribution patterns on protein coding genes, small rnas mapping to both introns and loci to which both sense and antisense small rnas mapped showed that both sets of small rnas have 5′-polyphosphate termini; suggesting that both transcripts may serve as template for small rna in order to determine whether small rna abundance patterns rnas from a non-virulent strain and found that small rnas mapped to genes in additionally, comparative analysis of small rna strand-specific rt-pcr detected transcripts in both directions at these loci account for strain-specific gene expression profiles of e. a manner consistent with their regulation of strain-specific virulence histolytica virulent and non-virulent strains, we sequenced small
 small rna mediated gene silencing is a well-conserved regulatory pathway. in the parasite entamoeba histolytica an endogenous rnai pathway exists, however, the depth and diversity of the small rna population remains unknown.  to characterize the small rna population that associates with e. histolytica argonaute-, we immunoprecipitated small rnas that associate with it and performed one full pyrosequencing run. data analysis revealed new features of the 27nt small rnas including the 5′-g predominance, distinct small rna distribution patterns on protein coding genes, small rnas mapping to both introns and exon-exon junctions, and small rna targeted genes that are clustered particularly in sections of genome duplication. characterization of genomic loci to which both sense and antisense small rnas mapped showed that both sets of small rnas have 5′-polyphosphate termini; strand-specific rt-pcr detected transcripts in both directions at these loci suggesting that both transcripts may serve as template for small rna generation. in order to determine whether small rna abundance patterns account for strain-specific gene expression profiles of e. histolytica virulent and non-virulent strains, we sequenced small rnas from a non-virulent strain and found that small rnas mapped to genes in a manner consistent with their regulation of strain-specific virulence genes.  we provided a full spectrum analysis for e. histolytica ago2-2 associated 27nt small rnas. additionally, comparative analysis of small rna populations from virulent and non-virulent amebic strains indicates that small rna populations may regulate virulence genes. 5′-polyphosphate small rnaargonautehigh-throughput pyrosequencingparasite 