BACKGROUND
a plethora of noncoding rnas , including small rnas that bind to proteins or base pair with target rnas, have been found to operate at all levels of gene regulation ranging from the control of enzymatic activity to the regulation of the initiation of transcription and translation  <cit> .
however, whole genome analyses of both prokaryotic and eukaryotic organisms have generally disregarded ncrna genes.
in bacteria, systematic searches for functional intergenic regions have led to the discovery of more than  <dig> bacterial trans-acting ncrnas of about  <dig> to  <dig> nucleotides mostly in e. coli  <cit> , but also in other pathogenic species  <cit> .
functional analysis of these ncrnas identified many of them as regulators of bacterial stress responses.
furthermore, the recent discovery of riboswitches  <cit>  and rna-based thermosensors  <cit>  in bacterial 5' untranslated regions  has enlarged the range of posttranscriptional control of gene expression.
in archaea, computational and experimental analysis lead to the identification and characterization of the homologues of the eukaryotic box c/d- and h/aca guide snornas, which are involved in modification and maturation of trnas and rrnas in crenarchaea and euryarchaea  <cit> .
rnomics studies have revealed the occurrence of stable antisense rnas and the expression of ncrnas from intergenic regions in sulfolobus solfataricus and archeoglobulus fulgidus  <cit> .
in silico searches using the pyrococcus furiosus and methanocaldococcus janashii genomes, predicted several novel ncrnas originating from intergenic regions  <cit> .
more recently, transcriptome analysis has revealed several dozen ncrnas in the halophilic euryarchaeon haloferax volcannii  <cit>  and more than two hundred in the methanogenic crenarchaeon methanosarcina mazeï  <cit> .
among the ncrnas in archeoglobulus fulgidus  <cit> , sulfolobus solfataricus, sulfolobus acidocaldarius  <cit>  and pyroccocus furiosus  <cit> , several correspond to ladder-like transcripts issued from repeated genomic sequences or crispr loci,  <cit> .
the crispr/cas defense system , identified in most archaeal genomes as well as many bacterial genomes, provides acquired immunity against viruses and plasmids by targeting nucleic acid in a sequence-specific manner  <cit> .
the anatomy of a crispr locus has been defined as an array of short direct repeats of  <dig> to  <dig> base pairs, often containing palindromic sequences  <cit> .
irrespective of the precise mechanism of the defensive action of crispr/cas systems, there is a consensus that transcription of the crispr cassette initiates in or near the leader sequence followed by processing by the cas proteins of the rna precursor into fragments  corresponding to the interval of the repeats  <cit> .
several stable rnas in the archaea, including the box c/d and h/aca guide rnas, the ribosomal rnas and rnase p, form ribonucleoprotein complexes  with the multifunctional l7ae protein that binds to the kink-turn   <cit>  or the related kink-loop  rna structural motif  <cit> .
these widespread motifs  <cit>  provide a platform for assembly of rnps or, in the case of the s-adenosylmethionine and lysine riboswitches, orient strands that base pair to form pseudoknots  <cit> .
the goal of this study was to identify and characterize novel ncrnas in pyrococcus abyssi, a thermococcal archaeon , which is one of the first archaea whose genome was sequenced  <cit> .
stable noncoding rnas that have been identified in p. abyssi are ribosomal rnas , rnase p , trnas , 7s rna , h/aca guide rnas  and c/d guide rnas .
most of these genes have a significantly higher  content compared to the rest of the genome, which is at rich.
the  content of the p. abyssi genome is 44% compared to 66% and 70% in rrna and trna genes, respectively.
considering the availability of several related thermococcal genomes and the at rich character of p. abyssi genome, we performed computational searches for novel ncrnas.
we clustered intergenic regions  based on primary and secondary structure features, and sequence conservation in other thermococcal genomes.
northern blotting showed that  <dig> out of the  <dig> selected igrs are transcribed.
additional primer extension and circular rapid amplification of cdna ends  experiments and in silico analysis showed that twelve of these transcripts have characteristics of regulatory ncrna: three are from crispr cassettes, three are from mrna 5'utrs and six are from intergenic regions.
altogether, this study allowed us to define two novel families of ncrna in p. abyssi, the cripsr and the 5'utr derived ncrnas.
RESULTS
detection of novel ncrnas
the pyrococcus abyssi igrs were screened using complementary approaches .
the first one, which takes into account specific characteristics of the p. abyssi genome, recovered  <dig> gc-rich regions in  <dig> igrs including  <dig> known ncrna genes and  <dig> novel regions.
the high number of known ncrna genes found by this approach confirms the interest in using at-rich genomes in computational search for ncrna genes.
the second comparative approach, which highlights sequence conservation between four closely related pyrococcal and thermococcal species, produced  <dig> regions similar to at least one region of another thermococcal genome.
the  <dig> regions cover  <dig> igrs of the p. abyssi genome.
based on alignment features,  <dig> regions were selected manually from both sets of candidates as described in the methods section.
fourteen regions encoding novel putative ncrnas were found by both approaches .
to refine our search,  <dig> regions predicted by both approaches were considered with regard to other criteria as described in the methods section including free energy of rna folding, presence of highly structured hairpins, and rna motifs such as k-turns and k-loops  <cit>  as well as similarities between the p. abyssi genome and all other available archaeal genomes.
altogether,  <dig> regions were selected as putative genes for ncrna .
to test whether all these regions express detectable transcripts, we performed northern blot analysis with strand-specific direct and reverse oligonucleotide probes , and rna extracted from p. abyssi in exponential, entry into stationary or stationary phase cultures.
signals were detected in  <dig> out of the  <dig> regions.
five regions generated transcripts up to  <dig> nt in length .
they were excluded from further analysis since they were likely to correspond to regions within polycistronic mrna.
we focused on the  <dig> regions transcribing rnas shorter than  <dig> nt.
seven regions corresponded to the h/aca rna genes recently annotated in the p. abyssi genome  <cit> .
the remaining  <dig> regions were clustered into four categories .
the first includes two crispr loci expressing three  <dig> nt ncrnas , the second includes four unique loci in the p. abyssi genome expressing rnas ranging in size from  <dig> to  <dig> nt, the third includes three repetitive loci conserved throughout thermococcal genomes expressing rnas ranging in size from  <dig> to  <dig> nt, and the fourth includes two repetitive loci specific to p. abyssi expressing rnas ranging in size from  <dig> to  <dig> nt.
more often than not, abundance of the expressed rnas was growth-regulated with the highest amounts detected in entry into stationary phase.
in many cases, several rna species arose from the same locus suggesting the processing of a primary transcript to secondary transcripts.
the positions indicate the beginning and the end of each predicted region.
the rna lengths are based on northern blot analysis.
the 5' ends of ncrnas were determined by primer extension experiments .
promotors were predicted based on search of entire  or partial  motif consensus located  <dig> to  <dig> nt  upstream of the 5' transcription start.
conservation among the  <dig> annotated thermococcal genomes: p. abyssi, p. horikoshii , p. furiosus , t. kodokarensis , t. sibiricus, t. gammatolerans and t. onnurineus.
prediction tools are denoted for each region: bias composition  and comparative analysis .
§ crispr  <dig> and  <dig> direct repeats are conserved.
* srk <dig> is annotated as ssca in p. furiosus  <cit> .
annotation and expression of p. abyssi crispr loci
the comparative computational screen selected the four crispr loci annotated recently in the p. abyssi genome  <cit> .
a careful sequence inspection of these cassettes revealed an erratic number of repeats and spacers in the p. abyssi crispr loci.
based on recent observations showing that new spacers are integrated mainly on the side proximal to the leader sequence of the crispr cassette and that degenerated direct repeats accumulate at the distal 3' end of the cassette  <cit> , we revised the annotation of the p. abyssi crisprs .
the crispr  <dig>  and crispr  <dig>  are composed of  <dig> and  <dig> direct repeats and spacers, respectively, with similar  <dig> nt direct repeat sequences.
the observation that the penultimate and ultimate 3' end-direct repeats are degenerate by one to six mismatches confirms the orientation proposed by the ucsc archaeal genome browser  <cit> , which differs from the orientation proposed by  <cit> .
the atypically short crispr  <dig> locus contains only four spacers with degenerate direct repeat sequences related to crispr  <dig> and  <dig>  the crispr  <dig> cassette encompassing eight spacers is distinguished by the sequence of its  <dig> nt direct repeats.
in order to position the degenerate direct repeat  at the end of the crispr cassette, as in the majority of the crispr loci  <cit> , we propose that crispr  <dig> is encoded by the reverse strand as opposed to previously reported annotations .
no signals were detected by northern blotting with strand-specific direct or reverse probes against sequence matching spacers  <dig> and  <dig> of crispr  <dig> and spacer  <dig> of crispr  <dig> suggesting that these loci are not transcribed .
this tentative conclusion was confirmed by more sensitive tests involving primer extension analysis to map 5' ends of the crispr precursors and to identify promoters .
in contrast, probes against sequences matching spacer  <dig> of crispr  <dig> and spacers  <dig> and  <dig> of crispr  <dig> allowed the detection of transcripts in all three growth phases .
the approximately  <dig> nt length of crispr-derived rnas, named hereafter cr1- <dig>  cr4- <dig> and cr4- <dig>  are of the size reported for crrnas corresponding to a spacer sequence and part of the direct repeat  <cit> .
moreover, primer extensions with total rna extracted from cells in entry-stationary phase  accurately identified their 5' ends , which correspond to the dicing site reported for the p. furiosus endoribonuclease cas  <dig>  <cit> .
since only single reverse transcription arrests are observed  even though multiple northern blot signals are detected , we propose that length heterogeneity results from partial or incomplete 3'end processing.
in conclusion, the crispr  <dig> and  <dig> cassettes are transcribed to produce small crrnas in p. abyssi.
no signals corresponding to precursor or other intermediates were detected by northern hybridization .
nevertheless, primer extensions with oligonucleotides hybridizing to the 5' junction of their respective first direct repeat resulted in the detection of an rna precursor and thus permitted the identification of the transcription starts of the crispr  <dig> and  <dig> rna precursors  .
similar experiments with specific primers of crispr  <dig> and  <dig> did not reveal any rna precursor confirming that crispr  <dig> and  <dig> are not expressed.
signals were not detected in northern hybridization with probes against the ultimate spacer  <dig> of crispr  <dig> and spacer  <dig> of crispr  <dig>  which harbor degenerate repeats.
these results suggest that the direct repeats are important for the processing and/or stability of the mature crrnas.
to identify transcription signals, we analyzed the  <dig> bp region upstream of the first direct repeat of each p. abyssi crispr cassette.
this analysis recognized conserved at rich motifs arguing for the presence of promoters.
the crispr  <dig> leader sequence has three at-rich and one a-rich element upstream of the first direct repeat, not corresponding to any typical p. abyssi consensus promoter sequence as defined in  <cit> .
in contrast, the crispr  <dig> and  <dig> cassettes have consensus promoter sequences  <dig> nt upstream of the experimentally determined 5' starts of precr <dig> and  <dig> .
two additional short elements at position + <dig> and - <dig> relative to the transcription starts were detected in crispr  <dig> and  <dig>  these sequences are also observed in other thermococcal crispr cassettes at the same distance from the first direct repeat .
it should be noted that the general organization of the crispr  <dig> leader is similar to those of crispr  <dig> and  <dig> except for the distance between the + <dig> a-stretch and the first direct repeat .
this feature might account for the absence of transcription of the crispr  <dig> locus.
expression of unique intergenic regions conserved among thermococcal genomes
four ncrnas were clustered in this category .
for three of them, srk <dig>  srk <dig> and srk <dig>  expression is detected at about the same intensity in all growth conditions whereas srk <dig> is clearly growth phase-regulated with little or no detectable rna in stationary phase .
remarkably, the genomic contexts of srk <dig>  srk <dig> and srk <dig> are similar, all being flanked by orfs transcribed in the same direction.
both, srk <dig> and srk <dig> cover predicted promoter sequences of the downstream annotated genes .
the respective 5' ends  determined by primer extension experiments  are at an acceptable distance from the predicted promoter elements  for srk <dig> and srk <dig>  and overlap a predicted tata box for srk <dig> .
no such sequence is found upstream of srk <dig> which might suggest that its transcription depends on the upstream gene pab <dig> since this region is predicted to form an operon that includes pab <dig>  except for srk <dig>  these ncrnas are extremely well conserved in sequence and structure throughout other thermococcal genomes .
the proposed rna secondary structures, based on rnafold and multiple alignment covariation analysis, highlight compensatory variations that maintain hairpin structures and, for srk <dig>  a potential pseudoknot structure .
only srk <dig> was predicted and validated in a previous comparative analysis in p. furiosus .
srk <dig> has a predicted secondary structure with a basal p1-stem with a high proportion of co-variations, an apical p <dig> stem conserved in sequence, and a 3' end sequence composed of a and u rich stretches that do not fold into a stable secondary structure .
altogether, these characteristics suggest that srk <dig>  srk <dig> and srk <dig> might have conserved cellular functions in the thermococcales.
expression of repetitive intergenic regions conserved among thermococcal genomes
among the three ncrnas clustered in this category , srk <dig> shows sequence similarities with two additional regions in p. abyssi  and three in p. horikoshii
.
sequence similarities preclude the design of specific probes to distinguish between srk <dig> and  <dig> .
northern blot signals provided evidence for a growth stage-specific transcription pattern with the most abundant species migrating about  <dig> nt.
these transcripts could arise from either of the loci harboring an upstream tata-like promoter  .
cr-rt-pcr experiments failed to detect primary transcripts, but suggested that antisense transcripts are expressed from the srk <dig> operon .
specific rna structures or rna modifications could have impeded the accurate amplification of the primary transcripts.
moreover, related sequences in the p. horikoshii genome are all clustered with crispr cassettes showing an organization comparable to that observed for the  <dig>  region.
all srk <dig> variants are atypical in that they contain multiple short polya stretches making rna folding into stable helical structures implausible.
the two other ncrnas of this category are srk <dig> and srk <dig> .
they were identified independently in our initial computational search and are part of a set of six similar genomic sequences in p. abyssi.
the similarities extend to all thermococcal genomes with four, three, two and one copies in p. furiosus, p. horikoshii, t. sibiricus and t. kodokaraensis, respectively .
the sequence similarities precluded the design of specific probes for each locus.
therefore, the signals detected in the northern blot in figure 3a could arise from any of the six repeated genomic regions.
in order to identify the transcribed regions, a cr-rt-pcr experiment was performed.
most clones corresponded to the  <dig> nt transcript with a majority matching srk <dig> and a minority srk <dig> .
they shared similar 5' ends and variable 3' ends.
in addition, a small percentage of clones corresponded to longer variants of srk <dig>  mapping a transcription initiation site  <dig> nt downstream a predicted bre/tata box promoter .
the shorter 5'-monophosphorylated srk <dig> transcript is likely to have arisen from maturation of the longer primary transcript.
interestingly, rnafold and multiple sequence alignments suggest a highly conserved rna secondary structure within the thermococcales .
many nucleotides, particularly in the p <dig> and p <dig> stems, support co-variations arguing for structural conservation.
the majority of the cloned cr-rt-pcr fragments contain sequences corresponding to the p <dig>  p <dig> and p <dig> stem-loops whereas a minority includes the p <dig> stem-loop in their 5' part .
the p <dig> stem-loop is unusual since its primary sequence is highly conserved throughout the thermococcales giving this structure not only a high propensity to form, but also suggesting an important cellular function.
inspection of the p2-loop of srk <dig> suggested that it forms a k-loop, which is consistent with the gelretardation of 5'end-labeled srk52-transcripts in presence of the recombinant p. abyssi l7ae protein .
the p3-loop is variable and can be extended into a stem structure for the two p. abyssi srk48-like sequences.
expression of repetitive intergenic regions specific to p. abyssi
amounts of the two ncrnas of this category, srkb and srkc, were highest upon entry into stationary phase .
because these loci  have significant sequence similarities to four other regions dispersed in the p. abyssi genome, we performed additional experiments to determine if the other loci were transcribed.
despite the sequence similarities, we could design oligonucleotide probes specific for each repetitive locus and perform additional northern blot probing , cr-rt-pcr experiments  and primer extension analyses .
from these data, the srkb and srkc loci were shown to be the only regions transcribed.
in order to identify the 5' and 3' ends of these transcripts, three distinct cr-rt-pcr experiments were carried out.
one pair of primers specific of the srkb locus  and two pairs of primers specific of the srkc locus  were chosen to characterize the overall rnas expressed at these loci.
the majority of the r <dig> clones correspond to transcripts from the srkb locus harboring an identical 5'end and a variable 3'end of which half ended between position  <dig> to  <dig> nt and half between position  <dig> to  <dig> nt .
the length of srkb rnas as determined by cr-rt-pcr, corresponds to the sizes of the transcripts detected in figure 5a.
moreover, sequence analysis revealed two typical promoter consensus sequences  <dig> nt and  <dig> nt upstream of the experimentally determined 5' end .
interestingly the 3' extremities of all the rna species are in the first  <dig> to  <dig> nucleotides of the pab <dig> orf.
additional primer extension analysis with a specific probe within pab <dig> suggests that a unique rna precursor is transcribed from this locus and subsequent processing events including 3' end trimming generate the different srkb rna species .
the r <dig> and r <dig> cr-rt-pcr analyses permitted the identification of three stable rna species from the srkc locus .
the two longer rna species of roughly  <dig> nt  and  <dig> nt  share exactly the same 5' extremity.
the 5' ends of the shorter 64/ <dig> nt rna species are positioned  <dig> nt downstream .
in addition, primary transcripts were distinguished from processed ones by omission of treatment of the rna preparations with tobacco acid pyrophosphatase  .
the 5' ends of 211/ <dig> nt-rna species are mostly triphosphorylated.
as in the case of srkb, a promoter sequence is found at an acceptable distance  from the 5' end .
rna species transcribed from the srkc locus could result from 5' and 3' end processing events of the primary 211/ <dig> nt long transcripts resulting in precise 5' monophosphorylated ends and variable 3' ends.
however, it should be noted that the 64/66nt rna species were not detected in northern blot analysis with specific probes  suggesting that they are in low abundance in our rna preparations.
in contrast to the srkb locus, the srkc locus is located in a long intergenic region.
the 3' end of pab <dig> n, the nearest orf, is located approximately  <dig> bp upstream of the 5' end of srkc .
as mentioned earlier, the srkb and srkc loci are part of a set of six similar sequences that had no counterparts in other thermococcal genomes and are therefore specific to p. abyssi.
consistent with their lack of expression, the bre/tata promoter sequences are missing in the other four other p. abyssi loci .
strikingly, one of these loci corresponds to the 3' end of the pab <dig> open reading frame.
this orf has been identified as a 'single orfb element' of the is605/is <dig> family of transposases  <cit> .
interestingly, in our initial computational characterization, srkb and srkc were predicted to fold into a structure with a k-turn rna motif.
to test if srkb and srkc form a k-turn, we performed gel retardation assays with recombinant p. abyssi l7ae .
this assay reveals at least two distinct ribonucleoprotein complexes  suggesting that more than one l7ae protein binds to the srkb and srkc rnas.
to elucidate these rna-protein interactions, footprint experiments using rnase t <dig> protection were performed with radioactively labeled  <dig> nt srkb transcript .
two sets of clustered guanosines are protected from rnase t <dig> digestion in the presence of l7ae, suggesting k-turns in stems p <dig>  and p <dig>  .
further support for the existence of kt <dig> and kt <dig> was obtained by mutations of the sheared ga base pairs, which are the hallmark of the k-turn.
in this case, gel retardation assays showed that the shift with recombinant p. abyssi l7ae was abolished .
sequence alignment  and rna-fold predictions support the rna secondary structure shown in figure 6c.
altogether, our data reveal that several transcripts expressed from the srkb and srkc loci have k-turn motifs that might be important for their cellular function.
another well-known rna structural motif, a loop e  <cit> , might form in the upper part of p <dig>  however, since there is no specific ligand that interacts with loop e, validation of this fold requires a detailed structural analysis.
it is interesting to note that the initiation codon for the pab <dig> is part of the p <dig> stem of srkb suggesting a functional link with the expression of the downstream pab <dig>
discussion
discovery of novel ncrnas by combined bioinformatic approaches
the rationale for carrying out a search of ncrnas in p. abyssi, the best studied thermococcale, was that with the exception of the box h/aca  <cit>  and the box c/d  <cit>  noncoding guide rna families, few thermococcal ncrna families have been described.
our study provides evidence for the growth-regulated expression of  <dig> novel ncrnas in the p. abyssi genome consisting of three crrnas from crispr loci, four ncrnas from unique loci conserved throughout thermococcales of which one appears to be a homologue of the p. furiosus scca rna  <cit> , three ncrnas from repetitive loci conserved throughout thermococcales, and two ncrnas from repetitive loci specific to p. abyssi.
since small proteins  of less than  <dig> amino acids have been largely disregarded in genome annotations  <cit> , we cannot exclude the possibility that the ncrnas identified here encode sproteins or peptides.
we therefore searched for open reading frames within the ncrna sequences starting with aug, gug or uug, and ending with uga, uag or uaa.
for several of the ncrnas, it was possible to find small orfs of  <dig> to  <dig> amino acids.
the only significant orf identified corresponds to the distal portion of a putative transposase cds .
we also analyzed the gc content of the ncrnas.
we found that it ranges from 33% to 66%.
this large spectrum is consistent with the gc content for known ncrnas .
since the computational analyses were restricted to intergenic regions, no cis-encoded antisense ncrna complementary to orfs, as identified in transcriptome analyses of sulfolobus solfataricus and methanosarcina mazei  <cit>  could be predicted.
below, experimental and genomic features of the new p. abyssi ncrna families are discussed.
processing of ncrnas
in this study, transcripts of different length were identified at one locus suggesting that rna processing occurs in p. abyssi.
the srkb, srkc, srk <dig> and srk <dig> primary transcripts with triphoshorylated 5' ends are processed into shorter 5' end monophosphorylated transcripts through maturation reactions .
for example, the 3' end heterogeneities observed for srkb, srkc, srk <dig> and srk <dig> rnas could arise from trimming of the primary transcript by the 3' to 5' exonucleolytic activity carried by the pyrococcus exosome  <cit> .
the 5' monophosphorylated rnas could result from endonucleolytic or pyrophosphohydrolytic activity.
previous examples of processing in pyrococcus were limited to trna processing by rnase p  <cit>  and crrna maturation by the cas  <dig> endonuclease  <cit> .
recent results suggest that the 5' to 3' exonucleolytic activity of rnase j homologues may also be involved in rna processing in euryarchaea and crenarchaea  <cit> .
crispr-derived rnas
our detection of crrnas is the first demonstration of the existence of an active crispr defense system in p. abyssi.
the crispr loci  <dig> and  <dig> appear to be expressed independently of growth phase in our experimental conditions.
in agreement with the findings in p. furiosus  <cit> , no antisense transcription of these crispr loci was detected  excluding the possibility that double strand rna intermediates are formed.
this contrasts with the report that rna is transcribed from the complementary spacer strand in s. acidocaldaricus  <cit> .
this difference could reflect a specific feature of the sulfolobales or a technical issue regarding the sensitivity of the northern blots analysis used in the studies of p. furiosus and p. abyssi.
based on our data, we propose a new annotation for the four p. abyssi crispr arrays that differs from earlier proposals .
the presence of a typical consensual promoter in the leader sequences of crispr  <dig> and crispr  <dig> correlates with the detection of crispr-derived rnas from these loci.
it has been reported that crispr cassettes expressing crrnas provide acquired immunity  <cit> .
it should be noted that short regions of spacers  <dig> and  <dig> of crispr  <dig>  are complementary to two coding regions of pav <dig>  a virus that infects p. abyssi   <cit> .
no other similarities were observed between crispr  <dig> and  <dig> spacer sequences and known p. abyssi mobile genetic elements  <cit> .
the silent crispr  <dig> and  <dig> loci differ from the expressed cassettes in several ways.
first, the crispr  <dig> locus has divergent direct repeat and leader sequences.
the cripsr  <dig> locus is atypical by its leader sequence, the reduced number of repeats, and the sequence degeneracy of its repeats, suggesting that it is a relic of an active crispr cassette as mentioned in  <cit> .
in general, crispr cassettes are physically linked to a cohort of conserved cas or cmr genes, in varying orientation and order, that encode crispr-associated proteins .
it is interesting to note that the only cas gene  in the vicinity of p. abyssi crispr cassettes is located upstream of crispr  <dig> .
in the p. abyssi genome, all the other genes encoding cas/cmr core proteins are grouped into an operon located far from any crispr locus .
further studies will be required to elucidate the mode of action and the dynamics of the crispr/cas system in p. abyssi.
5' utr-derived ncrnas
distinct rna species are not always indicative of independently synthesized rnas.
as revealed by race experiments and analysis of genomic context, several ncrnas  seem to derive from mrna leaders.
this class of ncrnas appears to originate from maturation of longer transcripts, as we demonstrated for srkb and propose for srk <dig>  srk <dig> and srk <dig>  stable rnas derived from rna leaders were initially identified in e. coli and some appear to correspond to riboswitch elements such as rfn and thi  <cit> .
it is now well established that 5'utrs can encompass transcription-termination signals involving riboswitch structures  <cit> .
in archaea, it has recently been suggested that riboswitches may also exist.
based on comparative genomics, crcb has been found in archaea and bacteria, and tpp and flp in euryarchaea  <cit> .
the crcb element was found within our set of  <dig> candidates , but ncrna corresponding to this element was not detected by northern blotting under the conditions employed in this study.
to date, no biological evidence showing gene regulation by riboswitches has been reported in archaea.
nevertheless, the conserved  <dig> nt srk <dig> shares several feature with the bacterial preq <dig> riboswitch  <cit> .
pab  <dig>  adjacent to srk <dig>  is predicted to encode an atpase with a queosine synthesis-like protein domain .
the growth-regulated srkb rna is transcribed from a locus adjacent to pab <dig>  which encodes a putative atpase.
in bacteria, the expression of several atpase-like proteins is controlled by cis-mechanisms involving the sam-i riboswitch.
it is interesting to note in listeria monocytogenes that this type of riboswitch also acts in trans as a small ncrna to regulate expression of the virulence factor prfa  <cit> .
moreover, our data strongly suggest that srkb has the propensity to form k-turn structures, which are found to be functional rna motifs involved in the assembly of ribonucleoprotein complexes and in the orientation of pseudoknot interactions in the aptamer structures of the sam-i and lysine riboswitches  <cit> .
therefore, srkb could have a specific function in the p. abyssi cell that involves the regulation of the expression of the putative atpase encoded by pab <dig>  we speculate that srk <dig> and srkb may be representative of a novel family of archaeal ncrnas produced by transcription attenuation or rna processing.
finally, the promoter-associated srk <dig> and srk <dig> could be related to the recently discovered ncrnas associated with transcription starts sites reported in higher eukaryotes  <cit> , in yeast  <cit>  and in salmonella enterica serovar typhi  <cit> .
this class of ncrnas is thought to interfere with transcription of the downstream promoter by cis-mediated occlusion or by a trans-mechanism.
transposase-related ncrnas
in this study, srk <dig>  srk <dig>  srkb and srkc ncrnas, which are transcribed from repetitive loci, were shown to be related to a cds annotated as an orphan orfb element of the is <dig> and is200/ <dig> family,  <cit> .
for example, in the case of srk <dig> and srk <dig>  similar sequences correspond to 3' end coding regions annotated as partial orfb-like is <dig> transposase in p. furiosus and t. sibiricus.
in bacteria, it has been shown that is <dig> transposition events are controlled by a complementary cis-encoded antisense ncrna  <cit> , but this seems unrelated to our observations since the ncrna sequences matched the sense sequence of the 3' end of the annotated orfs.
however, it is pertinent to note that a link between ncrnas and transposase orfs was already observed in sulfolobus solfataricus  <cit>  and in salmonella  <cit> .
moreover, in sulfolobus solfataricus  <cit>  it was shown that several small rnas linked to annotated transposase orfs could bind the multifunctional ribosomal protein l7ae through recognition of an rna kink turn as is the case for srkb, srkc and srk <dig>
CONCLUSIONS
two novel p. abyssi ncrna families, the cripsr and the 5' utr-related ncrnas are described in this study.
we certainly missed other p. abyssi ncrna families because the biased composition screen only identifies gc rich ncrnas.
this is the case for the box c/d guide rnas with low gc content.
nevertheless, the high number of known ncrnas including the box h/aca rnas found by this approach confirms its relevance in searching for ncrnas in at-rich genomes.
a limitation to this search, which is a bias due to misannotated orfs and therefore intergenic regions, could not be excluded since sequence signals for archaeal transcription and translation are not as well defined as their counterparts in bacteria.
the discovery of novel ncrnas by our combined computational approaches emphasizes the potential diversity of ncrnas in archaea, which could be enlarged by global rnomic approaches such as rnaseq technology.
this would provide a deeper insight into the p. abyssi ncrna world and help improve our knowledge of the specific roles of p. abyssi ncrnas and their relationship to the 5'utrs described in this study.

 a plethora of noncoding rnas, including small rnas that bind to proteins or base pair with target rnas, have been found to operate at all levels of gene regulation ranging from the control of enzymatic activity to the regulation of the initiation of transcription and translation. however, whole genome analyses of both prokaryotic and eukaryotic organisms have generally disregarded ncrna genes. in bacteria, systematic searches for functional intergenic regions have led to the discovery of more than bacterial trans-acting ncrnas of about to nucleotides mostly in e. coli, but also in other pathogenic species. functional analysis of these ncrnas identified many of them as regulators of bacterial stress responses. furthermore, the recent discovery of riboswitches and rna-based thermosensors in bacterial 5' untranslated regions has enlarged the range of posttranscriptional control of gene expression. in archaea, computational and experimental analysis lead to the identification and characterization of the homologues of the eukaryotic box c/d- and h/aca guide snornas, which are involved in modification and maturation of trnas and rrnas in crenarchaea and euryarchaea. rnomics studies have revealed the occurrence of stable antisense rnas and the expression of ncrnas from intergenic regions in sulfolobus solfataricus and archeoglobulus fulgidus. in silico searches using the pyrococcus furiosus and methanocaldococcus janashii genomes, predicted several novel ncrnas originating from intergenic regions. more recently, transcriptome analysis has revealed several dozen ncrnas in the halophilic euryarchaeon haloferax volcannii and more than two hundred in the methanogenic crenarchaeon methanosarcina mazeï. among the ncrnas in archeoglobulus fulgidus, sulfolobus solfataricus, sulfolobus acidocaldarius and pyroccocus furiosus, several correspond to ladder-like transcripts issued from repeated genomic sequences or crispr loci,. the crispr/cas defense system, identified in most archaeal genomes as well as many bacterial genomes, provides acquired immunity against viruses and plasmids by targeting nucleic acid in a sequence-specific manner. the anatomy of a crispr locus has been defined as an array of short direct repeats of to base pairs, often containing palindromic sequences. irrespective of the precise mechanism of the defensive action of crispr/cas systems, there is a consensus that transcription of the crispr cassette initiates in or near the leader sequence followed by processing by the cas proteins of the rna precursor into fragments corresponding to the interval of the repeats. several stable rnas in the archaea, including the box c/d and h/aca guide rnas, the ribosomal rnas and rnase p, form ribonucleoprotein complexes with the multifunctional l7ae protein that binds to the kink-turn or the related kink-loop rna structural motif. these widespread motifs provide a platform for assembly of rnps or, in the case of the s-adenosylmethionine and lysine riboswitches, orient strands that base pair to form pseudoknots. the goal of this study was to identify and characterize novel ncrnas in pyrococcus abyssi, a thermococcal archaeon, which is one of the first archaea whose genome was sequenced. stable noncoding rnas that have been identified in p. abyssi are ribosomal rnas, rnase p, trnas, 7s rna, h/aca guide rnas and c/d guide rnas. most of these genes have a significantly higher content compared to the rest of the genome, which is at rich. the content of the p. abyssi genome is 44% compared to 66% and 70% in rrna and trna genes, respectively. considering the availability of several related thermococcal genomes and the at rich character of p. abyssi genome, we performed computational searches for novel ncrnas. we clustered intergenic regions based on primary and secondary structure features, and sequence conservation in other thermococcal genomes. northern blotting showed that out of the selected igrs are transcribed. additional primer extension and circular rapid amplification of cdna ends experiments and in silico analysis showed that twelve of these transcripts have characteristics of regulatory ncrna: three are from crispr cassettes, three are from mrna 5'utrs and six are from intergenic regions. altogether, this study allowed us to define two novel families of ncrna in p. abyssi, the cripsr and the 5'utr derived ncrnas.  detection of novel ncrnas the pyrococcus abyssi igrs were screened using complementary approaches. the first one, which takes into account specific characteristics of the p. abyssi genome, recovered gc-rich regions in igrs including known ncrna genes and novel regions. the high number of known ncrna genes found by this approach confirms the interest in using at-rich genomes in computational search for ncrna genes. the second comparative approach, which highlights sequence conservation between four closely related pyrococcal and thermococcal species, produced regions similar to at least one region of another thermococcal genome. the regions cover igrs of the p. abyssi genome. based on alignment features, regions were selected manually from both sets of candidates as described in the methods section. fourteen regions encoding novel putative ncrnas were found by both approaches. to refine our search, regions predicted by both approaches were considered with regard to other criteria as described in the methods section including free energy of rna folding, presence of highly structured hairpins, and rna motifs such as k-turns and k-loops as well as similarities between the p. abyssi genome and all other available archaeal genomes. altogether, regions were selected as putative genes for ncrna. to test whether all these regions express detectable transcripts, we performed northern blot analysis with strand-specific direct and reverse oligonucleotide probes, and rna extracted from p. abyssi in exponential, entry into stationary or stationary phase cultures. signals were detected in out of the regions. five regions generated transcripts up to nt in length. they were excluded from further analysis since they were likely to correspond to regions within polycistronic mrna. we focused on the regions transcribing rnas shorter than nt. seven regions corresponded to the h/aca rna genes recently annotated in the p. abyssi genome. the remaining regions were clustered into four categories. the first includes two crispr loci expressing three nt ncrnas, the second includes four unique loci in the p. abyssi genome expressing rnas ranging in size from to nt, the third includes three repetitive loci conserved throughout thermococcal genomes expressing rnas ranging in size from to nt, and the fourth includes two repetitive loci specific to p. abyssi expressing rnas ranging in size from to nt. more often than not, abundance of the expressed rnas was growth-regulated with the highest amounts detected in entry into stationary phase. in many cases, several rna species arose from the same locus suggesting the processing of a primary transcript to secondary transcripts. the positions indicate the beginning and the end of each predicted region. the rna lengths are based on northern blot analysis. the 5' ends of ncrnas were determined by primer extension experiments. promotors were predicted based on search of entire or partial motif consensus located to nt upstream of the 5' transcription start. conservation among the annotated thermococcal genomes: p. abyssi, p. horikoshii, p. furiosus, t. kodokarensis, t. sibiricus, t. gammatolerans and t. onnurineus. prediction tools are denoted for each region: bias composition and comparative analysis. § crispr and direct repeats are conserved. * srk is annotated as ssca in p. furiosus. annotation and expression of p. abyssi crispr loci the comparative computational screen selected the four crispr loci annotated recently in the p. abyssi genome. a careful sequence inspection of these cassettes revealed an erratic number of repeats and spacers in the p. abyssi crispr loci. based on recent observations showing that new spacers are integrated mainly on the side proximal to the leader sequence of the crispr cassette and that degenerated direct repeats accumulate at the distal 3' end of the cassette, we revised the annotation of the p. abyssi crisprs. the crispr and crispr are composed of and direct repeats and spacers, respectively, with similar nt direct repeat sequences. the observation that the penultimate and ultimate 3' end-direct repeats are degenerate by one to six mismatches confirms the orientation proposed by the ucsc archaeal genome browser, which differs from the orientation proposed by. the atypically short crispr locus contains only four spacers with degenerate direct repeat sequences related to crispr and the crispr cassette encompassing eight spacers is distinguished by the sequence of its nt direct repeats. in order to position the degenerate direct repeat at the end of the crispr cassette, as in the majority of the crispr loci, we propose that crispr is encoded by the reverse strand as opposed to previously reported annotations. no signals were detected by northern blotting with strand-specific direct or reverse probes against sequence matching spacers and of crispr and spacer of crispr suggesting that these loci are not transcribed. this tentative conclusion was confirmed by more sensitive tests involving primer extension analysis to map 5' ends of the crispr precursors and to identify promoters. in contrast, probes against sequences matching spacer of crispr and spacers and of crispr allowed the detection of transcripts in all three growth phases. the approximately nt length of crispr-derived rnas, named hereafter cr1- cr4- and cr4- are of the size reported for crrnas corresponding to a spacer sequence and part of the direct repeat. moreover, primer extensions with total rna extracted from cells in entry-stationary phase accurately identified their 5' ends, which correspond to the dicing site reported for the p. furiosus endoribonuclease cas. since only single reverse transcription arrests are observed even though multiple northern blot signals are detected, we propose that length heterogeneity results from partial or incomplete 3'end processing. in conclusion, the crispr and cassettes are transcribed to produce small crrnas in p. abyssi. no signals corresponding to precursor or other intermediates were detected by northern hybridization. nevertheless, primer extensions with oligonucleotides hybridizing to the 5' junction of their respective first direct repeat resulted in the detection of an rna precursor and thus permitted the identification of the transcription starts of the crispr and rna precursors. similar experiments with specific primers of crispr and did not reveal any rna precursor confirming that crispr and are not expressed. signals were not detected in northern hybridization with probes against the ultimate spacer of crispr and spacer of crispr which harbor degenerate repeats. these results suggest that the direct repeats are important for the processing and/or stability of the mature crrnas. to identify transcription signals, we analyzed the bp region upstream of the first direct repeat of each p. abyssi crispr cassette. this analysis recognized conserved at rich motifs arguing for the presence of promoters. the crispr leader sequence has three at-rich and one a-rich element upstream of the first direct repeat, not corresponding to any typical p. abyssi consensus promoter sequence as defined in. in contrast, the crispr and cassettes have consensus promoter sequences nt upstream of the experimentally determined 5' starts of precr and. two additional short elements at position + and - relative to the transcription starts were detected in crispr and these sequences are also observed in other thermococcal crispr cassettes at the same distance from the first direct repeat. it should be noted that the general organization of the crispr leader is similar to those of crispr and except for the distance between the + a-stretch and the first direct repeat. this feature might account for the absence of transcription of the crispr locus. expression of unique intergenic regions conserved among thermococcal genomes four ncrnas were clustered in this category. for three of them, srk srk and srk expression is detected at about the same intensity in all growth conditions whereas srk is clearly growth phase-regulated with little or no detectable rna in stationary phase. remarkably, the genomic contexts of srk srk and srk are similar, all being flanked by orfs transcribed in the same direction. both, srk and srk cover predicted promoter sequences of the downstream annotated genes. the respective 5' ends determined by primer extension experiments are at an acceptable distance from the predicted promoter elements for srk and srk and overlap a predicted tata box for srk. no such sequence is found upstream of srk which might suggest that its transcription depends on the upstream gene pab since this region is predicted to form an operon that includes pab except for srk these ncrnas are extremely well conserved in sequence and structure throughout other thermococcal genomes. the proposed rna secondary structures, based on rnafold and multiple alignment covariation analysis, highlight compensatory variations that maintain hairpin structures and, for srk a potential pseudoknot structure. only srk was predicted and validated in a previous comparative analysis in p. furiosus. srk has a predicted secondary structure with a basal p1-stem with a high proportion of co-variations, an apical p stem conserved in sequence, and a 3' end sequence composed of a and u rich stretches that do not fold into a stable secondary structure. altogether, these characteristics suggest that srk srk and srk might have conserved cellular functions in the thermococcales. expression of repetitive intergenic regions conserved among thermococcal genomes among the three ncrnas clustered in this category, srk shows sequence similarities with two additional regions in p. abyssi and three in p. horikoshii. sequence similarities preclude the design of specific probes to distinguish between srk and. northern blot signals provided evidence for a growth stage-specific transcription pattern with the most abundant species migrating about nt. these transcripts could arise from either of the loci harboring an upstream tata-like promoter. cr-rt-pcr experiments failed to detect primary transcripts, but suggested that antisense transcripts are expressed from the srk operon. specific rna structures or rna modifications could have impeded the accurate amplification of the primary transcripts. moreover, related sequences in the p. horikoshii genome are all clustered with crispr cassettes showing an organization comparable to that observed for the region. all srk variants are atypical in that they contain multiple short polya stretches making rna folding into stable helical structures implausible. the two other ncrnas of this category are srk and srk. they were identified independently in our initial computational search and are part of a set of six similar genomic sequences in p. abyssi. the similarities extend to all thermococcal genomes with four, three, two and one copies in p. furiosus, p. horikoshii, t. sibiricus and t. kodokaraensis, respectively. the sequence similarities precluded the design of specific probes for each locus. therefore, the signals detected in the northern blot in figure 3a could arise from any of the six repeated genomic regions. in order to identify the transcribed regions, a cr-rt-pcr experiment was performed. most clones corresponded to the nt transcript with a majority matching srk and a minority srk. they shared similar 5' ends and variable 3' ends. in addition, a small percentage of clones corresponded to longer variants of srk mapping a transcription initiation site nt downstream a predicted bre/tata box promoter. the shorter 5'-monophosphorylated srk transcript is likely to have arisen from maturation of the longer primary transcript. interestingly, rnafold and multiple sequence alignments suggest a highly conserved rna secondary structure within the thermococcales. many nucleotides, particularly in the p and p stems, support co-variations arguing for structural conservation. the majority of the cloned cr-rt-pcr fragments contain sequences corresponding to the p p and p stem-loops whereas a minority includes the p stem-loop in their 5' part. the p stem-loop is unusual since its primary sequence is highly conserved throughout the thermococcales giving this structure not only a high propensity to form, but also suggesting an important cellular function. inspection of the p2-loop of srk suggested that it forms a k-loop, which is consistent with the gelretardation of 5'end-labeled srk52-transcripts in presence of the recombinant p. abyssi l7ae protein. the p3-loop is variable and can be extended into a stem structure for the two p. abyssi srk48-like sequences. expression of repetitive intergenic regions specific to p. abyssi amounts of the two ncrnas of this category, srkb and srkc, were highest upon entry into stationary phase. because these loci have significant sequence similarities to four other regions dispersed in the p. abyssi genome, we performed additional experiments to determine if the other loci were transcribed. despite the sequence similarities, we could design oligonucleotide probes specific for each repetitive locus and perform additional northern blot probing, cr-rt-pcr experiments and primer extension analyses. from these data, the srkb and srkc loci were shown to be the only regions transcribed. in order to identify the 5' and 3' ends of these transcripts, three distinct cr-rt-pcr experiments were carried out. one pair of primers specific of the srkb locus and two pairs of primers specific of the srkc locus were chosen to characterize the overall rnas expressed at these loci. the majority of the r clones correspond to transcripts from the srkb locus harboring an identical 5'end and a variable 3'end of which half ended between position to nt and half between position to nt. the length of srkb rnas as determined by cr-rt-pcr, corresponds to the sizes of the transcripts detected in figure 5a. moreover, sequence analysis revealed two typical promoter consensus sequences nt and nt upstream of the experimentally determined 5' end. interestingly the 3' extremities of all the rna species are in the first to nucleotides of the pab orf. additional primer extension analysis with a specific probe within pab suggests that a unique rna precursor is transcribed from this locus and subsequent processing events including 3' end trimming generate the different srkb rna species. the r and r cr-rt-pcr analyses permitted the identification of three stable rna species from the srkc locus. the two longer rna species of roughly nt and nt share exactly the same 5' extremity. the 5' ends of the shorter 64/ nt rna species are positioned nt downstream. in addition, primary transcripts were distinguished from processed ones by omission of treatment of the rna preparations with tobacco acid pyrophosphatase. the 5' ends of 211/ nt-rna species are mostly triphosphorylated. as in the case of srkb, a promoter sequence is found at an acceptable distance from the 5' end. rna species transcribed from the srkc locus could result from 5' and 3' end processing events of the primary 211/ nt long transcripts resulting in precise 5' monophosphorylated ends and variable 3' ends. however, it should be noted that the 64/66nt rna species were not detected in northern blot analysis with specific probes suggesting that they are in low abundance in our rna preparations. in contrast to the srkb locus, the srkc locus is located in a long intergenic region. the 3' end of pab n, the nearest orf, is located approximately bp upstream of the 5' end of srkc. as mentioned earlier, the srkb and srkc loci are part of a set of six similar sequences that had no counterparts in other thermococcal genomes and are therefore specific to p. abyssi. consistent with their lack of expression, the bre/tata promoter sequences are missing in the other four other p. abyssi loci. strikingly, one of these loci corresponds to the 3' end of the pab open reading frame. this orf has been identified as a'single orfb element' of the is605/is family of transposases. interestingly, in our initial computational characterization, srkb and srkc were predicted to fold into a structure with a k-turn rna motif. to test if srkb and srkc form a k-turn, we performed gel retardation assays with recombinant p. abyssi l7ae. this assay reveals at least two distinct ribonucleoprotein complexes suggesting that more than one l7ae protein binds to the srkb and srkc rnas. to elucidate these rna-protein interactions, footprint experiments using rnase t protection were performed with radioactively labeled nt srkb transcript. two sets of clustered guanosines are protected from rnase t digestion in the presence of l7ae, suggesting k-turns in stems p and p. further support for the existence of kt and kt was obtained by mutations of the sheared ga base pairs, which are the hallmark of the k-turn. in this case, gel retardation assays showed that the shift with recombinant p. abyssi l7ae was abolished. sequence alignment and rna-fold predictions support the rna secondary structure shown in figure 6c. altogether, our data reveal that several transcripts expressed from the srkb and srkc loci have k-turn motifs that might be important for their cellular function. another well-known rna structural motif, a loop e, might form in the upper part of p however, since there is no specific ligand that interacts with loop e, validation of this fold requires a detailed structural analysis. it is interesting to note that the initiation codon for the pab is part of the p stem of srkb suggesting a functional link with the expression of the downstream pab discussion discovery of novel ncrnas by combined bioinformatic approaches the rationale for carrying out a search of ncrnas in p. abyssi, the best studied thermococcale, was that with the exception of the box h/aca and the box c/d noncoding guide rna families, few thermococcal ncrna families have been described. our study provides evidence for the growth-regulated expression of novel ncrnas in the p. abyssi genome consisting of three crrnas from crispr loci, four ncrnas from unique loci conserved throughout thermococcales of which one appears to be a homologue of the p. furiosus scca rna, three ncrnas from repetitive loci conserved throughout thermococcales, and two ncrnas from repetitive loci specific to p. abyssi. since small proteins of less than amino acids have been largely disregarded in genome annotations, we cannot exclude the possibility that the ncrnas identified here encode sproteins or peptides. we therefore searched for open reading frames within the ncrna sequences starting with aug, gug or uug, and ending with uga, uag or uaa. for several of the ncrnas, it was possible to find small orfs of to amino acids. the only significant orf identified corresponds to the distal portion of a putative transposase cds. we also analyzed the gc content of the ncrnas. we found that it ranges from 33% to 66%. this large spectrum is consistent with the gc content for known ncrnas. since the computational analyses were restricted to intergenic regions, no cis-encoded antisense ncrna complementary to orfs, as identified in transcriptome analyses of sulfolobus solfataricus and methanosarcina mazei could be predicted. below, experimental and genomic features of the new p. abyssi ncrna families are discussed. processing of ncrnas in this study, transcripts of different length were identified at one locus suggesting that rna processing occurs in p. abyssi. the srkb, srkc, srk and srk primary transcripts with triphoshorylated 5' ends are processed into shorter 5' end monophosphorylated transcripts through maturation reactions. for example, the 3' end heterogeneities observed for srkb, srkc, srk and srk rnas could arise from trimming of the primary transcript by the 3' to 5' exonucleolytic activity carried by the pyrococcus exosome. the 5' monophosphorylated rnas could result from endonucleolytic or pyrophosphohydrolytic activity. previous examples of processing in pyrococcus were limited to trna processing by rnase p and crrna maturation by the cas endonuclease. recent results suggest that the 5' to 3' exonucleolytic activity of rnase j homologues may also be involved in rna processing in euryarchaea and crenarchaea. crispr-derived rnas our detection of crrnas is the first demonstration of the existence of an active crispr defense system in p. abyssi. the crispr loci and appear to be expressed independently of growth phase in our experimental conditions. in agreement with the findings in p. furiosus, no antisense transcription of these crispr loci was detected excluding the possibility that double strand rna intermediates are formed. this contrasts with the report that rna is transcribed from the complementary spacer strand in s. acidocaldaricus. this difference could reflect a specific feature of the sulfolobales or a technical issue regarding the sensitivity of the northern blots analysis used in the studies of p. furiosus and p. abyssi. based on our data, we propose a new annotation for the four p. abyssi crispr arrays that differs from earlier proposals. the presence of a typical consensual promoter in the leader sequences of crispr and crispr correlates with the detection of crispr-derived rnas from these loci. it has been reported that crispr cassettes expressing crrnas provide acquired immunity. it should be noted that short regions of spacers and of crispr are complementary to two coding regions of pav a virus that infects p. abyssi. no other similarities were observed between crispr and spacer sequences and known p. abyssi mobile genetic elements. the silent crispr and loci differ from the expressed cassettes in several ways. first, the crispr locus has divergent direct repeat and leader sequences. the cripsr locus is atypical by its leader sequence, the reduced number of repeats, and the sequence degeneracy of its repeats, suggesting that it is a relic of an active crispr cassette as mentioned in. in general, crispr cassettes are physically linked to a cohort of conserved cas or cmr genes, in varying orientation and order, that encode crispr-associated proteins. it is interesting to note that the only cas gene in the vicinity of p. abyssi crispr cassettes is located upstream of crispr. in the p. abyssi genome, all the other genes encoding cas/cmr core proteins are grouped into an operon located far from any crispr locus. further studies will be required to elucidate the mode of action and the dynamics of the crispr/cas system in p. abyssi. 5' utr-derived ncrnas distinct rna species are not always indicative of independently synthesized rnas. as revealed by race experiments and analysis of genomic context, several ncrnas seem to derive from mrna leaders. this class of ncrnas appears to originate from maturation of longer transcripts, as we demonstrated for srkb and propose for srk srk and srk stable rnas derived from rna leaders were initially identified in e. coli and some appear to correspond to riboswitch elements such as rfn and thi. it is now well established that 5'utrs can encompass transcription-termination signals involving riboswitch structures. in archaea, it has recently been suggested that riboswitches may also exist. based on comparative genomics, crcb has been found in archaea and bacteria, and tpp and flp in euryarchaea. the crcb element was found within our set of candidates, but ncrna corresponding to this element was not detected by northern blotting under the conditions employed in this study. to date, no biological evidence showing gene regulation by riboswitches has been reported in archaea. nevertheless, the conserved nt srk shares several feature with the bacterial preq riboswitch. pab adjacent to srk is predicted to encode an atpase with a queosine synthesis-like protein domain. the growth-regulated srkb rna is transcribed from a locus adjacent to pab which encodes a putative atpase. in bacteria, the expression of several atpase-like proteins is controlled by cis-mechanisms involving the sam-i riboswitch. it is interesting to note in listeria monocytogenes that this type of riboswitch also acts in trans as a small ncrna to regulate expression of the virulence factor prfa. moreover, our data strongly suggest that srkb has the propensity to form k-turn structures, which are found to be functional rna motifs involved in the assembly of ribonucleoprotein complexes and in the orientation of pseudoknot interactions in the aptamer structures of the sam-i and lysine riboswitches. therefore, srkb could have a specific function in the p. abyssi cell that involves the regulation of the expression of the putative atpase encoded by pab we speculate that srk and srkb may be representative of a novel family of archaeal ncrnas produced by transcription attenuation or rna processing. finally, the promoter-associated srk and srk could be related to the recently discovered ncrnas associated with transcription starts sites reported in higher eukaryotes, in yeast and in salmonella enterica serovar typhi. this class of ncrnas is thought to interfere with transcription of the downstream promoter by cis-mediated occlusion or by a trans-mechanism. transposase-related ncrnas in this study, srk srk srkb and srkc ncrnas, which are transcribed from repetitive loci, were shown to be related to a cds annotated as an orphan orfb element of the is and is200/ family,. for example, in the case of srk and srk similar sequences correspond to 3' end coding regions annotated as partial orfb-like is transposase in p. furiosus and t. sibiricus. in bacteria, it has been shown that is transposition events are controlled by a complementary cis-encoded antisense ncrna, but this seems unrelated to our observations since the ncrna sequences matched the sense sequence of the 3' end of the annotated orfs. however, it is pertinent to note that a link between ncrnas and transposase orfs was already observed in sulfolobus solfataricus and in salmonella. moreover, in sulfolobus solfataricus it was shown that several small rnas linked to annotated transposase orfs could bind the multifunctional ribosomal protein l7ae through recognition of an rna kink turn as is the case for srkb, srkc and srk  two novel p. abyssi ncrna families, the cripsr and the 5' utr-related ncrnas are described in this study. we certainly missed other p. abyssi ncrna families because the biased composition screen only identifies gc rich ncrnas. this is the case for the box c/d guide rnas with low gc content. nevertheless, the high number of known ncrnas including the box h/aca rnas found by this approach confirms its relevance in searching for ncrnas in at-rich genomes. a limitation to this search, which is a bias due to misannotated orfs and therefore intergenic regions, could not be excluded since sequence signals for archaeal transcription and translation are not as well defined as their counterparts in bacteria. the discovery of novel ncrnas by our combined computational approaches emphasizes the potential diversity of ncrnas in archaea, which could be enlarged by global rnomic approaches such as rnaseq technology. this would provide a deeper insight into the p. abyssi ncrna world and help improve our knowledge of the specific roles of p. abyssi ncrnas and their relationship to the 5'utrs described in this study. 
several of the novel ncrnas have sequence similarities to orphan orfb transposase elements. analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel p. abyssi ncrna families. based on rna secondary structure predictions and experimental results, we show that three of the twelve ncrnas include kink-turn rna motifs, arguing for a biological role of these ncrnas in the cell. furthermore, our results show that several of the ncrnas are subjected to processing events by enzymes that remain to be identified and characterized. the crispr family is comprised of crrnas expressed from two of the four p. abyssi crispr cassettes. using a combination of in silico and experimental approaches, we identified and characterized novel p. abyssi ncrnas transcribed from intergenic regions, ten of which are conserved throughout the thermococcales. this work proposes a revised annotation of crispr loci in p. abyssi and expands our knowledge of ncrnas in the thermococcales, thus providing a starting point for studies needed to elucidate their biological function.
 noncoding rna has been recognized as an important regulator of gene expression networks in bacteria and eucaryota. little is known about ncrna in thermococcal archaea except for the eukaryotic-like c/d and h/aca modification guide rnas.  using a combination of in silico and experimental approaches, we identified and characterized novel p. abyssi ncrnas transcribed from intergenic regions, ten of which are conserved throughout the thermococcales. several of them accumulate in the late-exponential phase of growth. analysis of the genomic context and sequence conservation amongst related thermococcal species revealed two novel p. abyssi ncrna families. the crispr family is comprised of crrnas expressed from two of the four p. abyssi crispr cassettes. the 5'utr derived family includes four conserved ncrnas, two of which have features similar to known bacterial riboswitches. several of the novel ncrnas have sequence similarities to orphan orfb transposase elements. based on rna secondary structure predictions and experimental results, we show that three of the twelve ncrnas include kink-turn rna motifs, arguing for a biological role of these ncrnas in the cell. furthermore, our results show that several of the ncrnas are subjected to processing events by enzymes that remain to be identified and characterized.  this work proposes a revised annotation of crispr loci in p. abyssi and expands our knowledge of ncrnas in the thermococcales, thus providing a starting point for studies needed to elucidate their biological function. 