BACKGROUND
members of the argonaute family of proteins have been identified as key players in small-rna-guided silencing pathways.
of these, proteins of the piwi clade are predominantly expressed in germ cells where they associate with a small rna population distinct from that of mirnas, as it has been shown initially for mammals and insects  <cit> .
some of the piwi-associated small rnas  are involved in regulating the expression of transposable elements, a function which is particularly important during genome activation in the germ line.
evidence for this specific role of pirnas comes from the studies in y  <cit> , zebrafish  <cit>  and mouse  <cit> .
although piwi-protein-associated small rnas have also been discovered in worm   <cit> , their function in this species is less clear.
post-transcriptional silencing of retrotransposon transcripts is achieved by a pirna amplification mechanism called ping-pong  <cit> .
briefly, pirnas that are generated by a so far unidentified mechanism and are complementary to retrotransposon transcripts guide piwi proteins to cleave these transcripts.
the position of the piwi-catalyzed cleavage is the bond between the nucleotides that have base-pairing interactions with nucleotides  <dig> and  <dig> of the primary pirna.
this cleavage defines the 5' end of a secondary pirna that is generated from the transposon transcript.
because a very high proportion of pirnas have a uridine  at the first position and because the complementarity between pirnas and targets is expected to be nearly perfect, secondary pirnas typically have adenosines at position  <dig>  which base-pairs with the u at the first position of the pirna  <cit> .
after the 3' end of the secondary pirna is generated through cleavage by a yet unknown nuclease and is subsequently 2'-o-methylated by the action of the hen- <dig> methyltransferase  <cit> , the secondary pirna is loaded into a piwi protein to guide the cleavage of a new primary pirna precursor.
this results in the production of pirnas from the same loci from which the initial pirnas were derived.
though the function of pirnas has been characterized in detail in only a few species, evidence for piwi protein-catalyzed cleavage and secondary pirna production  has been provided for vertebrates  <cit> , insects  <cit> , flatworms  <cit> , sea anemones and sponges  <cit> , suggesting an early origin of the ping-pong mechanism.
in mouse, pirnas are observed in the developing germ cells starting from the stage of g1-arrested gonocytes in the embryonic mouse up to the round spermatids in adult mouse  <cit> .
two classes of pirnas, with somewhat different characteristic lengths have been described  <cit> : pirnas which are ≈  <dig> nucleotides  in length are observed prior to meiosis, pre-natally and in the pre-pachytene stage, whereas a population of longer pirnas appear to take over around the pachytene stage.
evidence for secondary pirnas production has been found up to this point only in the prenatal and pre-pachytene pirna populations, which associate with the mili and miwi <dig> proteins  <cit> .
in contrast, pachytene pirnas, which associate with both mili and miwi, are thought to consist solely of primary pirnas  <cit> .
after initial studies defined the characteristic length of pirnas to be in the range of 23- <dig> nucleotides  <cit> , larger than that of mirnas, most studies of pirnas extracted and analyzed sequence reads whose length was in this range.
longer potential intermediates and longer or shorter by-products of the amplification loop, that could arise during the processing steps that generate both 5' and 3' ends of pirnas and could be in principle observed in the deep sequencing libraries have therefore not been captured in these studies.
because such products may shed light on the individual steps of pirna production and pirna-dependent targeting, we re-analyzed the few published libraries from mouse, rat and platypus that covered a broader range of small rna sizes .
we further generated and analyzed an additional library of small rnas from adult mouse testes.
RESULTS
signature of processing products at pirna loci
the library generated by robine et al.
<cit>  from adult mouse testes lysate, containing both pre-pachytene and pachytene pirnas, is one of the few publicly available libraries of small rnas that covers a relatively broad range of sequence lengths .
from the 9'936' <dig> sequences that mapped to the mouse genome, 6'144' <dig> sequences mapped uniquely and with no errors.
we focused on these 6'144' <dig> sequences and determined genome-wide the relative distances between the 5' coordinates of small rnas mapping to opposite strands of the chromosomes .
when two sequences from opposite strands have the same 5' coordinate, this distance is  <dig>  whereas the primary and secondary pirnas that are related through the ping-pong mechanism give a signature distance of  <dig> nucleotides .
to avoid our results being dominated by a few very abundantly sequenced reads, we counted the pairs of sense-antisense sequences as follows.
given a sense read with copy number cs and an antisense read with copy number ca we count towards the distance la - ls between the 5' end of the sense  and antisense  loci the smallest of the two copy numbers.
for example, at a locus where we observed  <dig> sequences starting on the sense strand at position ls and  <dig> sequences on the antisense strand at position la, we count  <dig> observed 'pairs' at distance la - ls.
an even more restrictive analysis, counting only the number of loci where a particular 5' distance occurred yielded the same results .
as expected, we detected a strong signal characteristic for pirna-directed targeting , i.e.
we found a relatively high frequency of sense-antisense pairs with a distance of  <dig> between the 5' ends of their loci.
surprisingly, we found that a  <dig> nt distance between 5' ends is also very common, though it has not been reported before.
in total, about 1'010' <dig> of the uniquely, perfectly-mapping sequences in the library contributed to those two peaks .
reasoning that previous studies that did not report the peak at  <dig> nt investigated relatively long sequences , we split the sequence set based on the length of the small rnas and repeated the analysis.
indeed, restricting the analysis to sequences longer than  <dig> nt  revealed only the peak at  <dig> nt .
the peak at  <dig>  only became apparent when we included sequences with a broader length distribution, because it corresponds to a pattern in which on one strand there is a long sequence, in the range of a prototypical pirna, and on the opposite strand a shorter sequence  .
furthermore, we found that the shorter sequence is almost always  <dig> nucleotides in length .
p <dig> and p <dig> processing patterns co-localize
by inspecting some examples of pairs whose 5' ends were at a distance of  <dig> nt we noticed that in the duplex that would be formed by these sequences, the pairing between a  <dig> nt-long sequence and the long pirna sequence would start at the 11th nt from the 5' end of the long sequence, as if the 3' end of the  <dig> nt-long sequence were defined by the piwi protein-dependent cleavage of the target rna, coupled perhaps with the generation of the secondary pirnas.
therefore, we decided to address systematically the question of whether the p <dig> and p <dig> processing patterns co-occurred.
for this purpose we anchored the regions of overlap between sense and antisense pairs that gave rise to the p <dig> on one hand and the p <dig> peaks on the other hand at the middle of the interval defined by the 5' ends of the sense-antisense pairs .
we then performed a genome-wide cross-correlation analysis of those positions  and obtained two symmetrical peaks at - <dig> and  <dig>  indicating that the middle of a p <dig> interval occurs preferentially either  <dig> nt downstream or  <dig> nt upstream of the middle of a p <dig> interval .
by separately analyzing the cases in which the short sequence of a p28-defining pair occurred on the plus  and on the minus strands , we established that the peak at - <dig> corresponded to cases in which the short sequence was on the positive strand and the peak at  <dig> to cases in which the short sequence was on the negative strand, as expected from the sketch shown in figure 2b.
thus, cross-correlation analysis reveals that sense-antisense pairs with a distance of  <dig> between 5' ends indeed co-occur with sense-antisense pairs with a distance of  <dig> between 5' ends.
this configuration, shown in figure 2b, is formed by two long pirna sequences from opposite strands that overlap by  <dig> nucleotides, characteristic for the ping-pong mechanism, and a  <dig> nt-long sequence which is located upstream of one of the two pirnas .
from the 7' <dig> genomic loci, where the p <dig> pattern occurred and the 13' <dig> loci where the p <dig> pattern was detected, 2' <dig> loci had exactly this triplex .
short by-products are generated during pirna targeting
to determine at what stage in pirna biogenesis or targeting cascade the short products are generated, we retrieved the genomic sequence of all 2' <dig> loci from the mouse testes lysate data set where the two pirnas together with the short by-product occurred.
one of the main characteristics of primary pirnas being their u bias  <cit>  we determined the frequency of the u nucleotide at position  <dig> of the long pirna sequences generated from these 2' <dig> loci.
while the pirnas that are located on the same strand as the 19-mers have a frequency of 44% u at position  <dig>  the pirnas on the opposite strand have a much higher frequency, namely 78%.
this suggests that the 19-mer is a product that arises during pirna-guided cleavage of target transcripts, whose 5' end is defined by a yet-unknown nuclease.
to gain further insight into how the 5' end of the 19-mer processing product is generated, we examined the relationship between the size of short products whose 3' end appears to be defined by a pirna-guided cleavage event and the size of the guiding pirna.
for this purpose we extracted all loci at which we observed a sense-antisense pair of sequences, with the 3' end of the shorter member of the pair being  <dig> nucleotides downstream of the 5' end of the longer sequence.
we then recorded the lengths of the long and short member of the pair and generated a contour plot of the frequency of occurrence of different pairs of lengths.
as shown in figure 3a, while the sequence of the guide pirna varies more widely in length, between  <dig> and  <dig> nt, the length of the short sequence is sharply peaked at  <dig> nt.
thus, it appears that the 5' end of the short processing product is defined with respect to the 5' end of the guide pirna, rather than with respect to the length of the guide pirna or of the duplex that this forms with its target.
by inspecting regions where the p <dig> patterns occur, we noticed that within broader genomic regions, the 19-mers were preferentially located on one strand , contrary to what one would expect in a population of cells in which the ping-pong process is on-going.
to systematically address the question of whether 19-mers occur interspersed on the two strands of a pirna locus or show a strand bias, we built a simple markov model with two states, t and b, corresponding to 19-mers on the plus strand  and of those with the 19-mer on the minus strand .
we then estimated transition probabilities pt→t , pt→b, pb→t and pb→b from our data .
here, the transition probability pt→b corresponds to the probability that a 19-mer on the bottom strand follows after a 19-mer on the top strand.
if the p <dig> sites exhibit no strand bias, we expect similar transition probabilities pt→t ≈ pt→b ≈ pb→t ≈ pb→b ≈  <dig> , whereas a strand-bias would result in a higher transition probabilities pt→t and pb→b, with pb→t and pt→b being smaller.
from our data we estimated pt→t and pb→b to be  <dig> , and pb→t and pt→b  <dig> .
this suggests that in the sample that we analyzed it was typically one of the strands from a pirna locus that generated the pirna target and the 19-mers, not both as one may expect from the ping-pong model, even though secondary pirnas were produced.
generation of short by-products is conserved across species
reasoning that a meaningful processing pattern would be conserved across species, we analyzed publicly available testes lysate data sets from rat and platypus  <cit> .
indeed, we found evidence for the p <dig> processing pattern both in rat and platypus , suggesting that conserved enzymatic processes generate the short by-products in species ranging from platypus to mouse.
on the contrary, we were not able to identify a p <dig> peak in the large data sets that were obtained from ips of various proteins from the fly pirna pathway  <cit>  , suggesting that the generation of 19-mers may be restricted to vertebrates.
furthermore, the rat data covered small rnas not only from testes but also from brain, liver, kidney, spleen and heart, giving us the opportunity to determine whether the short processing products are testes-specific.
indeed, similarly to the p <dig> pattern, the p <dig> pattern was testes-specific , as expected if these products were generated in the testes-specific pirna pathway.
given that the 19-mers are sufficiently abundant for their capturing in sequencing samples and that the process that generates them is conserved across species, one may hypothesize that, similarly to pirnas, they are stabilized and carry out an independent function.
pirnas are known to be stabilized through methylation of their 3' end  <cit> , and we therefore wondered whether the 19-mers also carry this modification.
to address this question, we incubated small rnas from adult mouse testes with sodium periodate  and subjected them to β-elimination  <cit> .
rnas that contain both 2' and 3' hydroxyls react with naio <dig> and are shortened by one nucleotide during β-elimination.
rnas that have only one unmodified hydroxyl, like pirnas that are 2'-o-methylated at their 3' end, do not react with naio <dig>  analysis of the β-eliminated data set revealed that the 19-mers appear to be shortened by the β-elimination process.
thus, the 19-mers are not stabilized through methylation of their 3' termini , and further studies will be required to establish whether or not they have a biological function.
discussion
it is unclear whether these 19-mers have a biological function or are simply by-products of pirna biogenesis.
if they were incorporated into piwi proteins, they would allow the ping-pong mechanism to move on the transcript, instead of being limited to the location defined by the primary pirna because the cleavage that would be guided by the 19-mer would occur at a different position in the transcript relative to the cleavage that is induced by the secondary pirna located immediately downstream of the 19-mer.
interestingly, we did detect the p <dig> peak in a library constructed from miwi protein immunoprecipitate  <cit> , suggesting that 19-mers may associate with miwi .
however, it remains to be established whether they do so as guide rnas or as by-products of pirna targeting that are transiently associated with piwi proteins.
the p <dig> peak is not as clearly apparent in the library constructed from the immunoprecipitate of the mili protein .
we also find a much smaller number of loci with triplex configuration - primary pirna, secondary pirna and the 19-mer upstream of the secondary pirna - in the mili data  compared to the miwi data , in spite of the fact that number of perfectly and uniquely mapping sequences that we analyzed was larger for mili  compared to miwi  .
this is unexpected, because previous studies implicated mili rather than miwi in secondary pirna production in mouse  <cit> .
although 19-mers do appear to associate with piwi proteins, the fact that they are not stabilized by 3' end methylation argues against their function as pirnas.
their size and the apparent lack of the 3' end modification would allow them to load into argonaute proteins and function in rnai-like silencing as short interfering rnas.
further analyses, including also immunoprecipitates of the ago proteins from testis will be necessary for testing this hypothesis.
this table summarizes the mapping statistics for the three libraries from robine et al.
<cit>  and the testes lysate library that we generated and then subjected to β-elimination.
CONCLUSIONS
a reanalysis of the pirna sequence reads in three mammalian species surprisingly revealed that the signature of pirna directed targeting and secondary pirna generation is accompanied by  <dig> nt long sequences.
because these sequences occur at what appear to be piwi-protein-mediated cleavage sites, we speculate that their 3' ends are generated during the same cleavage event that generates the 5' ends of the secondary pirnas.
because the distance between the 5' end of the pirna and the 5' end of the 19-mers is very precisely defined, our current hypothesis is that a 5'→3' exonuclease, which stops once it approaches the piwi protein complex , is responsible for the formation of the 5' ends .
thus, the 19-mers that we identified as products of pirna targeting yield new insights into this process.
furthermore, because we cannot currently define precisely what a pirna is , the frequently occurring p <dig> processing pattern can be used to identify loci where pirna directed targeting takes place.

 members of the argonaute family of proteins have been identified as key players in small-rna-guided silencing pathways. of these, proteins of the piwi clade are predominantly expressed in germ cells where they associate with a small rna population distinct from that of mirnas, as it has been shown initially for mammals and insects. some of the piwi-associated small rnas are involved in regulating the expression of transposable elements, a function which is particularly important during genome activation in the germ line. evidence for this specific role of pirnas comes from the studies in y, zebrafish and mouse. although piwi-protein-associated small rnas have also been discovered in worm, their function in this species is less clear. post-transcriptional silencing of retrotransposon transcripts is achieved by a pirna amplification mechanism called ping-pong. briefly, pirnas that are generated by a so far unidentified mechanism and are complementary to retrotransposon transcripts guide piwi proteins to cleave these transcripts. the position of the piwi-catalyzed cleavage is the bond between the nucleotides that have base-pairing interactions with nucleotides and of the primary pirna. this cleavage defines the 5' end of a secondary pirna that is generated from the transposon transcript. because a very high proportion of pirnas have a uridine at the first position and because the complementarity between pirnas and targets is expected to be nearly perfect, secondary pirnas typically have adenosines at position which base-pairs with the u at the first position of the pirna. after the 3' end of the secondary pirna is generated through cleavage by a yet unknown nuclease and is subsequently 2'-o-methylated by the action of the hen- methyltransferase, the secondary pirna is loaded into a piwi protein to guide the cleavage of a new primary pirna precursor. this results in the production of pirnas from the same loci from which the initial pirnas were derived. though the function of pirnas has been characterized in detail in only a few species, evidence for piwi protein-catalyzed cleavage and secondary pirna production has been provided for vertebrates, insects, flatworms, sea anemones and sponges, suggesting an early origin of the ping-pong mechanism. in mouse, pirnas are observed in the developing germ cells starting from the stage of g1-arrested gonocytes in the embryonic mouse up to the round spermatids in adult mouse. two classes of pirnas, with somewhat different characteristic lengths have been described: pirnas which are ≈ nucleotides in length are observed prior to meiosis, pre-natally and in the pre-pachytene stage, whereas a population of longer pirnas appear to take over around the pachytene stage. evidence for secondary pirnas production has been found up to this point only in the prenatal and pre-pachytene pirna populations, which associate with the mili and miwi proteins. in contrast, pachytene pirnas, which associate with both mili and miwi, are thought to consist solely of primary pirnas. after initial studies defined the characteristic length of pirnas to be in the range of 23- nucleotides, larger than that of mirnas, most studies of pirnas extracted and analyzed sequence reads whose length was in this range. longer potential intermediates and longer or shorter by-products of the amplification loop, that could arise during the processing steps that generate both 5' and 3' ends of pirnas and could be in principle observed in the deep sequencing libraries have therefore not been captured in these studies. because such products may shed light on the individual steps of pirna production and pirna-dependent targeting, we re-analyzed the few published libraries from mouse, rat and platypus that covered a broader range of small rna sizes. we further generated and analyzed an additional library of small rnas from adult mouse testes.  signature of processing products at pirna loci the library generated by robine et al. from adult mouse testes lysate, containing both pre-pachytene and pachytene pirnas, is one of the few publicly available libraries of small rnas that covers a relatively broad range of sequence lengths. from the 9'936' sequences that mapped to the mouse genome, 6'144' sequences mapped uniquely and with no errors. we focused on these 6'144' sequences and determined genome-wide the relative distances between the 5' coordinates of small rnas mapping to opposite strands of the chromosomes. when two sequences from opposite strands have the same 5' coordinate, this distance is whereas the primary and secondary pirnas that are related through the ping-pong mechanism give a signature distance of nucleotides. to avoid our results being dominated by a few very abundantly sequenced reads, we counted the pairs of sense-antisense sequences as follows. given a sense read with copy number cs and an antisense read with copy number ca we count towards the distance la - ls between the 5' end of the sense and antisense loci the smallest of the two copy numbers. for example, at a locus where we observed sequences starting on the sense strand at position ls and sequences on the antisense strand at position la, we count observed'pairs' at distance la - ls. an even more restrictive analysis, counting only the number of loci where a particular 5' distance occurred yielded the same results. as expected, we detected a strong signal characteristic for pirna-directed targeting, i.e. we found a relatively high frequency of sense-antisense pairs with a distance of between the 5' ends of their loci. surprisingly, we found that a nt distance between 5' ends is also very common, though it has not been reported before. in total, about 1'010' of the uniquely, perfectly-mapping sequences in the library contributed to those two peaks. reasoning that previous studies that did not report the peak at nt investigated relatively long sequences, we split the sequence set based on the length of the small rnas and repeated the analysis. indeed, restricting the analysis to sequences longer than nt revealed only the peak at nt. the peak at only became apparent when we included sequences with a broader length distribution, because it corresponds to a pattern in which on one strand there is a long sequence, in the range of a prototypical pirna, and on the opposite strand a shorter sequence. furthermore, we found that the shorter sequence is almost always nucleotides in length. p and p processing patterns co-localize by inspecting some examples of pairs whose 5' ends were at a distance of nt we noticed that in the duplex that would be formed by these sequences, the pairing between a nt-long sequence and the long pirna sequence would start at the 11th nt from the 5' end of the long sequence, as if the 3' end of the nt-long sequence were defined by the piwi protein-dependent cleavage of the target rna, coupled perhaps with the generation of the secondary pirnas. therefore, we decided to address systematically the question of whether the p and p processing patterns co-occurred. for this purpose we anchored the regions of overlap between sense and antisense pairs that gave rise to the p on one hand and the p peaks on the other hand at the middle of the interval defined by the 5' ends of the sense-antisense pairs. we then performed a genome-wide cross-correlation analysis of those positions and obtained two symmetrical peaks at - and indicating that the middle of a p interval occurs preferentially either nt downstream or nt upstream of the middle of a p interval. by separately analyzing the cases in which the short sequence of a p28-defining pair occurred on the plus and on the minus strands, we established that the peak at - corresponded to cases in which the short sequence was on the positive strand and the peak at to cases in which the short sequence was on the negative strand, as expected from the sketch shown in figure 2b. thus, cross-correlation analysis reveals that sense-antisense pairs with a distance of between 5' ends indeed co-occur with sense-antisense pairs with a distance of between 5' ends. this configuration, shown in figure 2b, is formed by two long pirna sequences from opposite strands that overlap by nucleotides, characteristic for the ping-pong mechanism, and a nt-long sequence which is located upstream of one of the two pirnas. from the 7' genomic loci, where the p pattern occurred and the 13' loci where the p pattern was detected, 2' loci had exactly this triplex. short by-products are generated during pirna targeting to determine at what stage in pirna biogenesis or targeting cascade the short products are generated, we retrieved the genomic sequence of all 2' loci from the mouse testes lysate data set where the two pirnas together with the short by-product occurred. one of the main characteristics of primary pirnas being their u bias we determined the frequency of the u nucleotide at position of the long pirna sequences generated from these 2' loci. while the pirnas that are located on the same strand as the 19-mers have a frequency of 44% u at position the pirnas on the opposite strand have a much higher frequency, namely 78%. this suggests that the 19-mer is a product that arises during pirna-guided cleavage of target transcripts, whose 5' end is defined by a yet-unknown nuclease. to gain further insight into how the 5' end of the 19-mer processing product is generated, we examined the relationship between the size of short products whose 3' end appears to be defined by a pirna-guided cleavage event and the size of the guiding pirna. for this purpose we extracted all loci at which we observed a sense-antisense pair of sequences, with the 3' end of the shorter member of the pair being nucleotides downstream of the 5' end of the longer sequence. we then recorded the lengths of the long and short member of the pair and generated a contour plot of the frequency of occurrence of different pairs of lengths. as shown in figure 3a, while the sequence of the guide pirna varies more widely in length, between and nt, the length of the short sequence is sharply peaked at nt. thus, it appears that the 5' end of the short processing product is defined with respect to the 5' end of the guide pirna, rather than with respect to the length of the guide pirna or of the duplex that this forms with its target. by inspecting regions where the p patterns occur, we noticed that within broader genomic regions, the 19-mers were preferentially located on one strand, contrary to what one would expect in a population of cells in which the ping-pong process is on-going. to systematically address the question of whether 19-mers occur interspersed on the two strands of a pirna locus or show a strand bias, we built a simple markov model with two states, t and b, corresponding to 19-mers on the plus strand and of those with the 19-mer on the minus strand. we then estimated transition probabilities pt→t, pt→b, pb→t and pb→b from our data. here, the transition probability pt→b corresponds to the probability that a 19-mer on the bottom strand follows after a 19-mer on the top strand. if the p sites exhibit no strand bias, we expect similar transition probabilities pt→t ≈ pt→b ≈ pb→t ≈ pb→b ≈, whereas a strand-bias would result in a higher transition probabilities pt→t and pb→b, with pb→t and pt→b being smaller. from our data we estimated pt→t and pb→b to be, and pb→t and pt→b. this suggests that in the sample that we analyzed it was typically one of the strands from a pirna locus that generated the pirna target and the 19-mers, not both as one may expect from the ping-pong model, even though secondary pirnas were produced. generation of short by-products is conserved across species reasoning that a meaningful processing pattern would be conserved across species, we analyzed publicly available testes lysate data sets from rat and platypus. indeed, we found evidence for the p processing pattern both in rat and platypus, suggesting that conserved enzymatic processes generate the short by-products in species ranging from platypus to mouse. on the contrary, we were not able to identify a p peak in the large data sets that were obtained from ips of various proteins from the fly pirna pathway, suggesting that the generation of 19-mers may be restricted to vertebrates. furthermore, the rat data covered small rnas not only from testes but also from brain, liver, kidney, spleen and heart, giving us the opportunity to determine whether the short processing products are testes-specific. indeed, similarly to the p pattern, the p pattern was testes-specific, as expected if these products were generated in the testes-specific pirna pathway. given that the 19-mers are sufficiently abundant for their capturing in sequencing samples and that the process that generates them is conserved across species, one may hypothesize that, similarly to pirnas, they are stabilized and carry out an independent function. pirnas are known to be stabilized through methylation of their 3' end, and we therefore wondered whether the 19-mers also carry this modification. to address this question, we incubated small rnas from adult mouse testes with sodium periodate and subjected them to β-elimination. rnas that contain both 2' and 3' hydroxyls react with naio and are shortened by one nucleotide during β-elimination. rnas that have only one unmodified hydroxyl, like pirnas that are 2'-o-methylated at their 3' end, do not react with naio analysis of the β-eliminated data set revealed that the 19-mers appear to be shortened by the β-elimination process. thus, the 19-mers are not stabilized through methylation of their 3' termini, and further studies will be required to establish whether or not they have a biological function. discussion it is unclear whether these 19-mers have a biological function or are simply by-products of pirna biogenesis. if they were incorporated into piwi proteins, they would allow the ping-pong mechanism to move on the transcript, instead of being limited to the location defined by the primary pirna because the cleavage that would be guided by the 19-mer would occur at a different position in the transcript relative to the cleavage that is induced by the secondary pirna located immediately downstream of the 19-mer. interestingly, we did detect the p peak in a library constructed from miwi protein immunoprecipitate, suggesting that 19-mers may associate with miwi. however, it remains to be established whether they do so as guide rnas or as by-products of pirna targeting that are transiently associated with piwi proteins. the p peak is not as clearly apparent in the library constructed from the immunoprecipitate of the mili protein. we also find a much smaller number of loci with triplex configuration - primary pirna, secondary pirna and the 19-mer upstream of the secondary pirna - in the mili data compared to the miwi data, in spite of the fact that number of perfectly and uniquely mapping sequences that we analyzed was larger for mili compared to miwi. this is unexpected, because previous studies implicated mili rather than miwi in secondary pirna production in mouse. although 19-mers do appear to associate with piwi proteins, the fact that they are not stabilized by 3' end methylation argues against their function as pirnas. their size and the apparent lack of the 3' end modification would allow them to load into argonaute proteins and function in rnai-like silencing as short interfering rnas. further analyses, including also immunoprecipitates of the ago proteins from testis will be necessary for testing this hypothesis. this table summarizes the mapping statistics for the three libraries from robine et al. and the testes lysate library that we generated and then subjected to β-elimination.  a reanalysis of the pirna sequence reads in three mammalian species surprisingly revealed that the signature of pirna directed targeting and secondary pirna generation is accompanied by nt long sequences. because these sequences occur at what appear to be piwi-protein-mediated cleavage sites, we speculate that their 3' ends are generated during the same cleavage event that generates the 5' ends of the secondary pirnas. because the distance between the 5' end of the pirna and the 5' end of the 19-mers is very precisely defined, our current hypothesis is that a 5'→3' exonuclease, which stops once it approaches the piwi protein complex, is responsible for the formation of the 5' ends. thus, the 19-mers that we identified as products of pirna targeting yield new insights into this process. furthermore, because we cannot currently define precisely what a pirna is, the frequently occurring p processing pattern can be used to identify loci where pirna directed targeting takes place. 
the piwi protein-associated small rnas guide piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. this suggests that the processing of the 5' product of pirna-guided cleavage occurs while the pirna target is engaged by the piwi protein. cleavage of the retrotransposon transcript defines at the same time the 5' end of a secondary pirna that will in turn guide a piwi protein to a primary pirna precursor, thereby amplifying primary pirnas. although several studies provided evidence that this mechanism is conserved among metazoa, how the process is initiated and what enzymatic activities are responsible for generating the primary and secondary pirnas are not entirely clear. here we analyzed small rnas from three mammalian species, seeking to gain further insight into the mechanisms responsible for the pirna amplification loop. they will enable more accurate identification of pirna loci in deep sequencing data sets.
 the pirna pathway operates in animal germ lines to ensure genome integrity through retrotransposon silencing. the piwi protein-associated small rnas guide piwi proteins to retrotransposon transcripts, which are degraded and thereby post-transcriptionally silenced through a ping-pong amplification process. cleavage of the retrotransposon transcript defines at the same time the 5' end of a secondary pirna that will in turn guide a piwi protein to a primary pirna precursor, thereby amplifying primary pirnas. although several studies provided evidence that this mechanism is conserved among metazoa, how the process is initiated and what enzymatic activities are responsible for generating the primary and secondary pirnas are not entirely clear.  here we analyzed small rnas from three mammalian species, seeking to gain further insight into the mechanisms responsible for the pirna amplification loop. we found that in all these species pirna-directed targeting is accompanied by the generation of short sequences that have a very precisely defined length, nucleotides, and a specific spatial relationship with the guide pirnas.  this suggests that the processing of the 5' product of pirna-guided cleavage occurs while the pirna target is engaged by the piwi protein. although they are not stabilized through methylation of their 3' ends, the 19-mers are abundant not only in testes lysates but also in immunoprecipitates of miwi and mili proteins. they will enable more accurate identification of pirna loci in deep sequencing data sets. 