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HYPOTHESIS

piRNAs derived from ancient viral processed pseudogenes
as transgenerational sequence-specific immune memory
in mammals
NICHOLAS F. PARRISH,1,8,9 KAN FUJINO,1,8,11 YUSUKE SHIROMOTO,2,10 YUKA W. IWASAKI,3 HONGSEOK HA,4
JINCHUAN XING,4 AKIKO MAKINO,1,5 SATOMI KURAMOCHI-MIYAGAWA,2 TORU NAKANO,2
HARUHIKO SIOMI,3 TOMOYUKI HONDA,1,6 and KEIZO TOMONAGA1,6,7
1

Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
Department of Pathology, Medical School and Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan3
Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
4
Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA
5
Center for Emerging Virus Research, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
6
Department of Tumor Viruses, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
7
Department of Mammalian Regulatory Network, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan
2

ABSTRACT
Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse
transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species
with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that
EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known
to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs
independently some 25–40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more
often than expected by chance alone (P = 8 × 10−3–6 × 10−8). Three of the seven human EBLNs fall within annotated piRNA
clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise
to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters
containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived
from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent
with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that
retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune
memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.
Keywords: endogenous viral elements; retrotransposon; piRNA; CRISPR/Cas; paleovirology

INTRODUCTION
Retroviruses integrate DNA copies of their RNA genome
into that of their host, transferring genetic information
in a direction not predicted by the central dogma (Crick1958). Understanding how these gene transfers influence
the immunologic distinction between host “self ” and viral
8

These authors contributed equally to this work.
Present address: Section of Surgical Sciences, Vanderbilt University
School of Medicine, Nashville, TN 37232, USA
10
Present address: Laboratory of Gene Expression and Regulation, The
Wistar Institute, Philadelphia, PA 19104, USA
11
Present address: Department of Microbiology II, School of Veterinary
Medicine, Azabu University, Kanagawa 252-5201, Japan
Corresponding authors: nicholas.parrish@vanderbilt.edu, tomonaga@
virus.kyoto-u.ac.jp
Article published online ahead of print. Article and publication date are at
http://www.rnajournal.org/cgi/doi/10.1261/rna.052092.115.
9“nonself ” is of practical importance. Interestingly, several
host genes derived from retroviruses limit infection by related
viruses (Ikeda and Odaka 1983; Hainaut et al. 1990; Muraet al. 2004; Aswad and Katzourakis 2012; Fujino et al.
2014; Yap et al. 2014), demonstrating that lateral geneflow from viruses to their vertebrate hosts can mediatetransgenerational immune memory; all known examples
are mediated by production of dominant negative proteins. RNA-mediated transgenerational antiviral immunity,
while common via the CRISPR/Cas system in prokaryotes© 2015 Parrish et al. This article is distributed exclusively by the RNA
Society for the first 12 months after the full-issue publication date (seehttp://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it isavailable under a Creative Commons License (Attribution-NonCommercial
4.0 International), as described at http://creativecommons.org/licenses/bync/4.0/.

RNA 21:1–13; Published by Cold Spring Harbor Laboratory Press for the RNA Society

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Parrish et al.

(Sorek et al. 2008), has thus far been reported in only one
metazoan, Caenorhabditis elegans (Rechavi et al. 2011). Immunity through the CRISPR/Cas system requires lateraltransfer of genetic information: short nonself nucleic acid“spacers” are integrated into specialized genomic arraysof repeated sequences. Transcripts from these specialized
self loci are processed into ribonucleoprotein complexes capable of degrading nonself targets on the basisof Watson–Crick base-pairing with spacer RNA (Barrangou et al. 2007). A conceptually similar system mediatedby PIWI-interacting RNAs (piRNAs) appears broadly
conserved in metazoans (Grimson et al. 2008), yet the confirmed targets are largely limited to endogenous transposable
elements.
In addition to retroviruses, RNA-only viruses have also
given rise to sequences in many metazoan genomes, although
the specific mechanisms involved in this horizontal RNA-toDNA information flow are less clear (Belyi et al. 2010; Horieet al. 2010; Katzourakis and Gifford 2010). We have studied
one class of these sequences, called endogenous bornaviruslike nucleoprotein elements (EBLNs), because they are theonly riboviral endogenous elements known in humans.
Homo sapiens EBLNs (hsEBLNs) contain poly(dA:dT) tracts,
recognizable transcription start sites, and are flanked by target-site duplications, strongly suggesting that they represent
viral mRNA integrated by a retrotransposon (Esnault et al.
2000; Belyi et al. 2010). These EBLNs were integrated ∼40
million years ago, coincident with the peak of host processed
pseudogene formation by a similar mechanism (Zhang et al.
2003). Because species with EBLNs appear relatively protected against modern day bornaviruses (Belyi et al. 2010), negative-strand RNA viruses that can cause neurological disease(Tomonaga et al. 2002), we questioned if they could influence antiviral immunity like some endogenous retroviralelements.
HsEBLN-1 and -2 contain long open reading frames(ORFs) with the potential to code for proteins of 366 and
225 amino acids, respectively. In cell culture experiments,
overexpression of bornaviral nucleoprotein prevents infection, presumably because a specific stoichiometry of replication complex components is critical (Geib et al. 2003). Thus
overexpression of EBLN-encoded proteins could potentiallyhave prevented ancient bornaviral replication. Indeed, we recently showed that the Ictidomys tridecemlineatus genome
contains an EBLN that was integrated more recently than human EBLNs, shares over 75% of amino acids in common
with some extant avian bornaviruses, and can block bornaviral replication when overexpressed in human cells (Fujino
et al. 2014). However, most EBLNs in primates and rodents
have disrupted ORFs (Horie et al. 2010), and there is no evidence of selection to maintain the ORFs of EBLNs in primates (Kobayashi et al. 2011). Thus most EBLNs either had
no function, had a protein-coding function that has been
lost, or perhaps had a function not related to encoding aprotein.
2

RNA, Vol. 21, No. 10

We recently observed that all seven human EBLNs are expressed as RNAs, some exclusively in the adult testis (KSofuku, N Parrish, T Honda, and K Tomonaga, in prep.).
Because the native promoter sequence is not mobilized during pseudogene formation, the probability of seven out ofseven randomly chosen host processed pseudogenes being
expressed is low; by the highest estimates, only about onethird of host processed pseudogenes are transcribed (Zheng
et al. 2007; Guo et al. 2014). This led us to investigate the possibility that mammalian EBLNs encode antiviral RNAs.
The previously noted similarities between the piRNA pathway and the CRISPR/Cas immune system (Karginov and
Hannon 2010) made piRNAs an attractive candidate. Analogous to CRISPR guide RNA, piRNA, in complex with a
PIWI-clade argonaute protein partner, target transposons
for transcriptional and post-transcriptional silencing (Siomi
and Kuramochi-Miyagawa 2009; Ishizu et al. 2012). Similar
to CRISPR arrays, piRNA precursor molecules are transcribed from discrete loci (“piRNA clusters”) that cover asmall percentage of the total genome (Lau et al. 2006; Aravin
et al. 2007). It is thought that these loci act as “traps,” in thesense that nucleic acid elements transposing into them willsubsequently be silenced (Malone and Hannon 2009). PiRNAs are most abundant in the germline, consistent with arole in genome defense, yet some piRNA pathway components are detectable in somatic cells where their functional
relevance is unclear. Here we show that multiple EBLNs, integrated independently in two mammalian lineages at least 20
million years ago, give rise to piRNAs, and present evidence
consistent with selection for EBLNs that integrated into piRNA-generating loci.

RESULTS
Rodent EBLNs give rise to small RNA withcharacteristics of piRNAs
We noted EBLN-derived piRNAs while examining rodent
EBLN sequences using a web-based genome browser (Karolchik et al. 2014). Several piRNAs identified in reports initiallydescribing piRNAs (Aravin et al. 2006; Girard et al. 2006; Lauet al. 2006) overlap with Mus musculus and Rattus norvegicus
EBLNs (Table 1). Most piRNAs are generated from genomic
loci that are unannotated (Girard et al. 2006), as are most
EBLNs. Thus to better ascertain the abundance and diversityof EBLN-derived small RNAs, we sequenced small RNAs
from the testis of a 6-wk-old mouse. Small RNAs with sequence characteristics of primary piRNAs, namely a length
over 26 nucleotides and enrichment of 5′ uridine, mapped
to three of the five mouse EBLNs (mmEBLN-3 through -5)(Fig. 1A). The abundance of small RNAs mapped to
mmEBLN-5 was highest, with thousands of reads mapped
to some regions of this sequence. The abundance of small
RNAs mapped to mmEBLN-3 and -4 was lower, yet above

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Hypothesis: CRISPR/Cas-like immunity in mammals

TABLE 1. GenBank annotated piRNA derived from rodent EBLNs

Mus musculus
EBLN-5

EBLN-3

Accession

Name

Coordinates

Length5′ Nucleotide

DQ549815
DQ548337
DQ689528
DQ700150
DQ703437
DQ724667
DQ706487
DQ711296
DQ725925
DQ698401
DQ551691
NA

mmu_piR_003423
mmu_piR_003006
mmu_piR_012394
mmu_piR_019537
mmu_piR_021794
mmu_piR_036414
mmu_piR_023855
mmu_piR_027183
mmu_piR_037293
mmu_piR_018347
mmu_piR_004100
mmu_piR_038731

chr9:54093758–54093787
chr9:54093761–54093790
chr9:54093806–54093834
chr9:54093814–54093842
chr9:54093845–54093874
chr9:54093868–54093898
chr18:67195283–67195313
chr18:67195514–67195543
chr18:67195670–67195699
chr18:67195905–67195932
chr18:67195934–67195965
chr18:67196113–67196142

30
30
29
29
30
31
31
30
30
28
32
30

U
U
U
U
U
U
U
U
U
A
U
U

rno_piR_009248
rno_piR_008949
rno_piR_003427
rno_piR_007299
rno_piR_038475
rno_piR_026877
rno_piR_015474
rno_piR_001654
rno_piR_029961
rno_piR_031873
rno_piR_027737
rno_piR_019953

chr8:57747085–57747114
chr8:57747093–57747122
chr8:57747095–57747125
chr8:57747130–57747158
chr8:57747226–57747254
chr8:57747268–57747296
chr8:57747276–57747304
chr8:57747283–57747311
chr8:57747287–57747316
chr8:57747336–57747365
chr8:57747349–57747379
chr18:63553931–63553956

30
30
31
29
29
29
29
29
30
30
31
26

U
U
U
U
U
U
U
U
U
U
U
U

Rattus norvegicus
EBLN-5
DQ622271
DQ621647
DQ609231
DQ617717
DQ767477
DQ750254
DQ732898
DQ605336
DQ754944
DQ757774
DQ751527
EBLN-3
DQ739763(U) Uridine, (A) adenosine.

the level of most genomic loci. In contrast, no small RNAs
mapped to mmEBLN-1 and -2. Examining the small RNAs
mapped to the 100 kb flanking these EBLNs revealed thatmmEBLN-3 and -5 were within loci with small RNAs abundantly mapped to a single genomic strand, while mmEBLN-4
was near the end of such a locus (Fig. 1B). Notably, allpiRNA-like small RNAs mapped to the antisense strand relative to the hypothetical ancient bornaviral N mRNA whose
integration was responsible for EBLN formation. This experiment suggested that small RNAs with characteristics ofpiRNAs were generated from three of five mmEBLN loci,and that these EBLNs were in or near piRNA clusters. Thus
we examined publically available sequence data sets andpiRNA cluster annotations to further evaluate this possibility.

Rodent EBLNs are enriched in pachytene
piRNA clusters
A piRNA is defined as a small RNA in complex with a PIWI
protein, thus immunoprecipitation of intact ribonucleoproteins is required to unambiguously confirm the identity of
any RNA molecule as a piRNA. Thus we first analyzed sequences from a comprehensive analysis of murine piRNAbiogenesis (Li et al. 2013a). Consistent with our initial experiment, small RNAs mapped to three of five mmEBLNs(mmEBLN-3, -4, and -5) (Table 2). These RNAs ranged inlength from 25 to 31 nucleotides (nt) and >95% contained
uridine as the 5′ nucleotide (Fig. 1C). All were antisense
relative to the proposed ancient bornaviral mRNA, consistent
with the potential to post-transcriptionally silence such
mRNA (Reuter et al. 2011). As expected of piRNAs derived
from a primary piRNA transcript, and as opposed to secondary piRNAs generated during the so-called “ping-pong” amplification cycle that occurs prenatally in mice (Beyret et al.
2012), there was no enrichment of adenosine at the 10th nucleotide position (Wang et al. 2014). Li et al. (2013b) defined
417 piRNA precursor transcripts from which over 95%
of mature murine piRNA are derived. Together these sequences cover only 0.28% of the mouse genome. Pachytene
piRNA transcripts, which give rise to the predominant classof piRNAs found in adult testis, cover only 0.08% of the genome. Thus a short genomic range chosen at random has anexceedingly low likelihood of being within such a transcript.
However, two of the five mmEBLNs (mmEBLN-3 and -5) arewithin pachytene piRNA transcripts. Moreover, the piRNA
precursors containing these two EBLNs give rise to the third
and 13th highest density of mapped piRNAs from the adultmouse testis (Lau et al. 2006). mmEBLN-4 is within 40 kbof the 3′ termini of an annotated pachytene piRNA transcript.
To estimate the probability of this apparent enrichment of
EBLNs within piRNA-generating genomic loci, we calculatedwww.rnajournal.org

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Parrish et al.

FIGURE 1. Murine EBLNs give rise to piRNA. (A) Testis small RNAs map to murine EBLNs. Borna disease virus (BDV), nucleoprotein (N)gene, and homologous murine EBLNs (1–5 as numbered) are depicted. Numbers atop each marker represent the EBLN length as defined bytranslated amino acid homolog to BDV determined in Arensburger et al. (2011). Numbers below each marker indicate the genomic coordinatesof the murine EBLNs. Uniquely mapped small RNAs are shown for EBLN-3 through -5; reads antisense to the predicted ancient bornaviralmRNA are plotted in green below each gene marker, sense reads in blue above. The y-axis indicates the number of mapped reads. (B)mmEBLN-3 through -5 are in or near clusters of mapped small RNAs. Small RNA reads matching the 50 kb upstream and downstream
from mmEBLN-3 through -5 are shown. Reads mapping to the 5′ to 3′ strand are shown in blue above the line and those mapped to the opposite
strand are shown in green below the line. The y-axis indicates the number of uniquely mapped reads and is arbitrarily truncated at 1000 or 5000
reads. (C) mmEBLN-mapped reads have characteristics of piRNAs. The nucleotide composition of small RNAs mapped to murine EBLN-3
through -5 is shown, with each base colored as indicated. Bases 1, 10, 26, and 31 are numbered. The y-axis indicates the percentage nucleotides
at each position of all mapped reads corresponding to each base.

the cumulative binomial distribution of an event with a probability 0.0028 occurring twice or more in, as there are five recognized EBLNs, five trials (P = 8 × 10−5).
Considering the possibility that this enrichment was perhaps limited to a single species, we also examined the genomeof the laboratory rat (Rattus norvegicus). All EBLNs are insyntenic loci in mice and rats (Horie et al. 2013), suggestingthat these EBLNs were integrated between 20 and 30 million
years ago into a common ancestor shared by these species(Horie et al. 2013). PiRNA clusters are generally well conserved between mice and rats in terms of their genomic context (Assis and Kondrashov 2009), but not at the level ofpiRNA sequence (Aravin et al. 2006). As primary piRNA precursor transcripts have not been defined in the rat, we exam4

RNA, Vol. 21, No. 10

ined piRNA cluster annotations to determine if EBLNderived piRNAs would be expected to be made in this species.
Indeed, both orthologous EBLNs within piRNA clusters inmice were annotated in rat piRNA clusters (Girard et al.
2006; Lau et al. 2006), and rnEBLN-4 was also annotatedwithin a cluster in one study (Girard et al. 2006). As in themouse, a low percentage of the overall rat genome gives
rise to piRNAs and is annotated as a piRNA clusters(0.18% in the more inclusive annotation). The probabilityof this degree of enrichment of rnEBLNs in piRNA-generating loci occurring by chance, calculated as above, ranges from6 × 10−8 to 1 × 10−6 based on the annotation used (Fig. 2A).
In summary, three out of the five EBLNs in mice and rats giverise to piRNAs. As we are limited to detecting EBLNs thatcluster 85g; chr4:122158319–
122199619

cluster 6f; chr8:57725000–
57777000piRNA clusterh–
ID 1201; chr3:73061025–
73070092
ID 2841; chr9:37086668–
37092983––
ID 3562; chr11:75001149–
75023707–

chr4:122161250–122161697 (+)

chr8:57747079–57747381 (+)

Coordinates (strand)

chr10:22208838–22209999 (−)
chr3:73062206–73063317 (+)

chr9:37086679–37087837 (+)

chr17:4570963–4572180 (+)
chr1:235117490-235118542 (−)
chr11:75011855–75012971 (−)

chr10:94534165–94535277 (−)

rnEBLN-4

rnEBLN-5

hsEBLN-1
hsEBLN-2

hsEBLN-3

hsEBLN-4
hsEBLN-5
hsEBLN-6

hsEBLN-7

chr7:19830726–19831493 (−)–––
cluster 14f; chr18:63622000–
63658000

chr10:92784712–92785688 (−)
chr10:82624298–82625334 (+)
chr18:63553420–63554192 (+)

rnEBLN-1
rnEBLN-2
rnEBLN-3

cjEBLN-1

piRNA cluster

Coordinates (strand)

piRNA clusteri–
9-qA5.3-24188.1;
chr9:54054980–54097630

chr6:87894228–87895035 (+)
chr9:54093749–54093919 (+)

mmEBLN-4mmEBLN-5

Coordinates (strand)––
18-qE1-36451.1;
chr18:67189100–67226114

piRNA precursor transcripta

chr11:103490037–103490965 (−)
chr11:93851147–93851916 (−)
chr18:67195203–67196180 (+)

Coordinates (strand)

mmEBLN-1
mmEBLN-2
mmEBLN-3

EBLN

TABLE 2. piRNA abundance from rodent and primate EBLNs

Ancestral: human ID 5251;
chr18:11631026–11717899 (marmoset
chr13:45297101–45343400)
Ancestral: human ID 1264;
chr3:128540550–128603591(marmoset chr15:68183501–
68217900)
Ancestral: human ID 4384;
chr15:51535076–51605926 (marmoset
chr10:28388101–28440800)

Ancestral: human ID 4384;
chr15:51535076–51605926 (marmoset
chr10:28388101–28440800)

Ancestral: human ID 5251;
chr18:11631026–11717899 (marmoset
chr13:45297101–45343400)

piRNA cluster evolutionb

5.28
RPKMi
Complementary to
N mRNA
0

0.07
0
3.35

0

RPKMh
Complementary to
N mRNA
0.12
0

ND

ND

RPKM
Complementary to
N mRNA00
ND

5.84
603.4800144.11

Complementary to
N mRNAd

www.rnajournal.org

Continued

Complementary to viralgenomic RNA
0

0

0
0
0

1.71

Complementary to viralgenomic RNA
0
0.68

0

0

Complementary to viralgenomic RNA
0
0
0

0
0

0
0
0

Complementary to viralgenomic RNAe

RPKMc

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Hypothesis: CRISPR/Cas-like immunity in mammals

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6RNA, Vol. 21, No. 10

chr15:49481896–49483299 (+)
chr1:138998195–138998975 (+)
GL25135.1:14259–15765 (+)
chr19:45134664–45135692 (−)
chr11:63682272–63683372 (−)
chr12:81451040–81452148 (−)

Coordinates (strand)––––
chr11:63677801–63689900–

piRNA precursor transcripta

piRNA cluster evolutionb

0
0
0
0
15.81
1.23

Complementary to
N mRNAd

0
0
0
0
0
0

Complementary to viralgenomic RNAe

RPKMc

(mmEBLN) Mus musculus EBLN; annotated based on mm9 (strand equivalent to hypothetical ancient mRNA). (rnEBLN) Rattus norvegicus EBLN; annotated based on RGSC3.4. (hsEBLN)
Homo sapiens EBLN; annotated based on GRCh38/hg38. (cjEBLN) Callithrix jacchus EBLN; annotated based on cj3.2. (ND) Not determined.
a
From Li et al. (2013a), Supplemental Table 2 (0.28% of the genome annotated as a piRNA cluster).
b
From Assis and Kondrashov (2009).
c
Mapped from Li et al. (2013b); GEO accession number GSM1096587.
dAccording to hypothetical ancient bornaviral N mRNA.
e
As a negative-strand RNA virus, the bornavirus genomic strand is the reverse complement of the mRNA strand.
fFrom Lau et al. (2006) (0.106% annotated as a piRNA cluster).
g
From Girard et al. (2006) (0.18% annotated as a piRNA cluster).
h
From Ha et al. (2014) (2.4% annotated as a piRNA cluster at RPKM cutoff of >1) liftover converted to GRCh38/hg38.
i
From Hirano et al. (2014) (0.12% annotated as a piRNA clus).

cjEBLN-2
cjEBLN-2
cjEBLN-4
cjEBLN-5
cjEBLN-6
cjEBLN-7

EBLN

TABLE 2. Continued

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Hypothesis: CRISPR/Cas-like immunity in mammals(Hirano et al. 2014). As in rodents, thepiRNAs produced by these EBLNs are annon-piRNA
tisense to the proposed ancient bornaviralencoding genome
mRNA. Experimental differences could
explain why hsEBLN-2 and -3 give rise
3 EBLNs within
3 EBLNs within
piRNA clusters
piRNA clusters
piRNA clusters
to piRNA-like small RNAs in the testis
while the syntenic marmoset EBLNs
EBLNs
in the marmoset do not: the marmoset
2 EBLNs outside
4 EBLNs outside
sequences represent bona fide piRNAs
piRNA clusters
piRNA clusters
immunoprecipitated with MARWI, while
FIGURE 2. EBLNs are enriched within piRNA clusters. (A) The genome of Rattus norvegicus is the human testis small RNA was bulkdepicted. Non-piRNA-generating sequences in the genome (purple) and piRNA clusters (red, isolated and enriched for 2′ -O-methylat0.18%, as annotated in Girard et al. (2006) are drawn to scale. EBLNs are depicted as blue dots ed small RNAs (Kirino and Mourelatos
and are drawn larger than scale to allow visualization. The probability of the observed enrichment,
estimated as described in the text, is 6 × 10−8. (B) The genome of Homo sapiens is depicted as 2007). Alternatively, this could reflect
above, with 2.4% of the genome annotated as piRNA cluster. The probability of the observed en- loss of piRNA production from cjEBLNrichment is 4.5 × 10−4.
2 and -3 loci in the ∼35 million years since
the marmoset and human lineages dihave maintained recognizable homology with modern borverged. In any case, these observations confirm that indenaviruses, we do not assume that the existing five rodent
pendent EBLN integrations are enriched within piRNA- or
EBLNs are the only sequences derived from ancient bornapiRNA-like RNA-generating loci in two mammalian lineages.
viruses that ever entered the genomes of these species.
The probability of this occurring due to chance, approximatedNonetheless, given the relative sparsity of piRNA-generating
as the union of the most likely probabilities when each lineage
genomic loci, we can reasonably exclude the hypothesis thatis considered independently, is very low (P = 6.4 × 10−7).
rodent EBLNs were integrated at random and maintained
neutrally thereafter.
Rodent EBLNs were integrated into existingpiRNA clusters
piRNAs are generated from primate EBLNs
Precisely what defines a piRNA precursor transcript assuch is currently unclear (Vourekas et al. 2015), as are the deEBLNs are found in many vertebrate genomes (Belyi et al.
terminants of innate immune recognition of bornaviral nu2010), and similar to retrotransposon insertions, are predictcleic acids (Martin et al. 2011). In invertebrates, piRNAed to be essentially free of homoplasy. About 40 million years
like RNAs can be generated from viral sequences (Wu et al.
ago, EBLNs were integrated into a common primate ancestor
2010; Morazzani et al. 2012; Léger et al. 2013), and knockprior to the divergence of the strepsirrhine and haplorrhine
down of piRNA pathway components has a proviral effect inprimates (Horie et al. 2010). Accordingly, these primate
mosquito cells (Schnettler et al. 2013) and flies (Zambon
EBLNs were each integrated into different loci than rodent
et al. 2006). Viral piRNA biogenesis in these invertebrates
EBLNs. We hypothesized that if EBLN-derived piRNAs wereis assumed to be due to an interaction between viral genomic
functional, independent EBLN integrations with piRNAor transcript RNAs with PIWI proteins and piRNA pathway
generating capacity may have evolved convergently in thesecomponents in the absence of an integrated DNA intermeditwo lineages. Thus we determined if piRNA-like RNAs or
ate. Moreover, in Aedes aegypti cells distinct PIWI proteinspiRNAs were made from the seven EBLNs in human andare required for piRNA biogenesis from viral precursors
marmoset genomes, respectively, as deep sequencing of small
compared with endogenous transposon precursors. Thus
RNAs from adult testes from both species has recently beenpresumably some feature of at least some viral nucleic acids
performed (Ha et al. 2014; Hirano et al. 2014). Similar tois sufficient to determine their recognition for processing
pachytene piRNAs derived from adult mouse testes, theinto piRNA-like molecules. The RNA-binding proteins inpiRNA populations in the testis of adult marmosets are provolved in piRNA precursor processing (e.g., MOV10L1)
cessed from primary piRNA precursor transcripts rather
may share common specificities with RNA-binding proteins
than ping-pong amplification. In humans, three EBLNs
involved in recognizing infectious viruses (e.g., MOV10(hsEBLN-2, -3, and -6) were found to be within annotated[Wang et al. 2010]), thus it is conceivable that integration
piRNA clusters (Fig. 2B; total 2.4% annotated, P = 4.5 ×of an EBLN into an existing transcriptional unit could have10−4). hsEBLN-7 was also found to give rise to piRNA-like
RNA, yet was shorter than the arbitrary length cutoff used tobeen responsible for that transcript’s definition as a piRNA
define clusters (Table 2). In the marmoset, one EBLN
precursor. If this were the case, the convergent evolution de(cjEBLN-6) was within an annotated cluster (total 0.12% anscribed above could have arisen in a direct, mechanistic fashnotated, P = 0.008) and cjEBLN-7 also gave rise to piRNAs asion. We can reject this possibility in several instances, as somesociated with the marmoset PIWI-like 1 protein MARWI
piRNA clusters are syntenic between primates and rodents
A

R. norvegicus

B

H. sapienswww.rnajournal.org

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Parrish et al.

ancestral
non-piRNA
encoding genome

rodentpiRNA cluster

primateEBLN-3, -4, -5

FIGURE 3. Rodent EBLNs were integrated into existing piRNA clusters. The genome of the common rodent/primate ancestor is depicted,
with sequence predicted to generate piRNAs shown in red and thenon-piRNA encoding genome in purple. After the divergence of rodents
and primates, an EBLN (blue) was integrated into this pachytene piRNA
precursor transcriptional unit. This pattern of evolution occurred foreach EBLN currently found in rodent genomes, suggesting that EBLN
sequences were not themselves required for piRNA-generating capacity
to their surrounding piRNA cluster sequences.

(Hirano et al. 2014). Indeed, rodent EBLN-3 and -5 are within large intergenic piRNA clusters also present in humans,
as is the rat piRNA cluster containing EBLN-4 (Table 2;
Fig. 3). The marmoset EBLNs generating bona fide piRNAs(cjEBLN-6 and -7) are within the 3′ untranslated regionsof genes (NEU3 and TBC1D12) that also generate piRNAs
in the mouse, albeit much less abundantly (Robine et al.
2009). Thus at least some EBLNs were integrated into preexisting piRNA clusters. Beyond this, we cannot exclude an influence of EBLN integration on the life/death evolutionary
dynamics of piRNA clusters (Assis and Kondrashov 2009).

DISCUSSION
Two models could account for the convergent evolution ofpiRNA-generating EBLNs in two mammalian lineages: onein which EBLNs were preferentially integrated within
piRNA clusters, and another in which EBLNs integrated randomly and those within piRNA clusters were positively selected. We cannot exclude the first model, but consider it
unlikely based on the distribution of other nucleic acids mobilized by retrotransposons: Among the over 10,000 and 5000
GENCODE-annotated (Harrow et al. 2012) processed pseudogenes in the human and mouse genomes, respectively,
few are within piRNA clusters (Ha et al. 2014; Hirano et al.
2014; Watanabe et al. 2015). Furthermore, LINE-1-mobilized sequences in cultured cells (Berry et al. 2006), tumors(Cooke et al. 2014; Tubio et al. 2014), and mice bearing anengineered LINE-1 element (An et al. 2006) are not preferentially targeted to piRNA clusters. Finally, recent processed
pseudogene (Abyzov et al. 2013) and retrotransposon insertions in human genomes are not enriched within piRNAgenerating loci (Y Zhang and P Gerstein, pers. comm.).
Nonetheless, experiments to determine if, under certain circumstances, nonself nucleic acids are preferentially trapped
within piRNA clusters are warranted (Kawaoka et al. 2013).
8

RNA, Vol. 21, No. 10

Enrichment of EBLNs within piRNA-generating loci intwo mammalian lineages is consistent with natural selection (Fig. 4). EBLN-derived piRNAs may have fortuitously
silenced a host gene shared by these lineages, or multiple
EBLNs could have hitchhiked with linked alleles. Anotherparsimonious hypothesis is that EBLN-derived piRNAs
silenced bornavirus and thus protected against death or
reduced fecundity due to bornaviral infection, reminiscentof transposon (Khurana et al. 2011) or errantiviral (Prud’homme et al. 1995) resistance after sequences from these elements are integrated into Drosophila piRNA clusters. Suchan effect is feasible and consistent with current models of
the mammalian piRNA system if ancient bornaviruses weretransmitted vertically via the gamete. Indeed, the ancient bornaviruses giving rise to EBLNs infected germ cells; otherwise
a noncanonical mechanism of gene transfer from soma togermline, in violation of Weismann’s law, was responsible
for EBLN formation (Pittoggi et al. 2006). Of note, modern
bornaviruses can be transmitted vertically (Okamoto et al.
2003; Kerski et al. 2012). EBLN integration into piRNA clusters could thus have resulted in viral silencing in germ cells,
similar to the transgene silencing observed after insertionof identical sequences into piRNA clusters (Yamamoto
et al. 2013). As transcriptional silencing via repressive chromatin modification, rather than post-transcriptional silencing, appears the dominant mechanism of piRNA-mediatedsilencing, it is notable that modern bornaviruses, unlike
most RNA viruses, replicate in the nucleus and interact
directly with chromatin (Matsumoto et al. 2012). Further,while an antiviral effect of RNA interference has been detected in certain mammalian systems (Li et al. 2013b), it isconsidered to have been largely superseded in mammalianantiviral innate immunity by type I interferons (Cullen
et al. 2013; Cullen 2014). However, overexpression of interferon α prevents germ-cell development in mice and its receptor is not expressed on pachytene spermatocytes (Satieet al. 2011), perhaps censoring this antiviral mechanism in
the germline (Pare and Sullivan 2014).
PiRNA-like RNAs and/or PIWI proteins have been described in primate pluripotent cells (Marchetto et al. 2013),
human hematopoietic cells (Sharma et al. 2001; Cichocki
et al. 2010), and some other somatic cells including neurons(Lee et al. 2011; Yan et al. 2011; Rajasethupathy et al. 2012), a
critical target cell of modern bornaviruses. Notably neurons,
like germ cells, are permissive to retrotransposition (Upton
et al. 2015) and relatively nonresponsive to type I interferons(Lin et al. 2013; Kreit et al. 2014). Transcripts of the murine
PIWI-clade partner of most pachytene piRNAs (MIWI) canbe detected in a distribution overlapping highly BDV-susceptible cells in the cerebellum, dentate gyrus, and olfactory bulb
(Lein et al. 2007; Ackermann et al. 2010). Thus EBLN-derived piRNAs could potentially have protected stem orsomatic cells like neurons from bornavirus-induced pathology, although more work is required to assess the feasibilityof this model. A class of piRNA-like RNAs derived from theDownloaded from rnajournal.cshlp.org on August 17, 2015 - Published by Cold Spring Harbor Laboratory Press

Hypothesis: CRISPR/Cas-like immunity in mammals

X
transcript
reversionanamnestic
piRNA silencing

EBLN
enrichment
in piRNAclusters:

re-exposure

4.5x10-4
Homo sapiens

L1 ORF2p?

EBLN

Strepsirrhini
common ancestor

ancient bornavirus
germline infection

bornavirus mRNA
(P)TGS?
bornavirus-anamnestic
piRNA or piRNA-like RNA

8x10-3Callithrix jaccus

40 myapiRNA cluster
1x10-6

non-piRNA
encoding genome
Rattus norvegicus

20-30 mya

rodent/primate
common ancestor

Murodia
common ancestor

8x10-5bornavirus-mediatedselection?
Mus musculus

FIGURE 4. Conceptual model of mammalian EBLN formation and selection. A model chromosome of a common rodent/primate ancestor is shownat left. The non-piRNA encoding genome is shown in purple, piRNA clusters in red, and EBLN integrations are indicated in blue. Multiple sequencesfrom ancient bornaviruses were integrated into the germline of individuals of the indicated lineages via transcript reversion at the time written ([mya]millions of years ago). The likely candidate for such activity in the primate lineage is the LINE-1 ORF2p, encoded by the host genome. We hypothesizethat natural selection, perhaps mediated by differential survival or fecundity when animals with EBLNs faced subsequent bornavirus infection, could
be responsible. One potential mechanism for this selective advantage, piRNA or piRNA-like RNA-guided viral gene silencing via post-transcriptionalor transcriptional gene silencing ([P]TGS), is depicted. The observation that EBLNs are enriched within piRNA clusters in multiple species of eachlineage is improbable in the absence of selection, and an approximation of this probability is listed for each species.

same genomic loci that give rise to pachytene piRNAs, yetwith different genetic requirements for their biogenesis,
have recently been described in a variety of somatic cell types(Ortogero et al. 2014). In addition, dicer-dependent endogenous small interfering RNAs are produced from some piRNA
clusters (Watanabe et al. 2008). Thus production of piRNAs
from EBLNs in the testis could correlate with productionof other, more relevant, RNA molecules from these locielsewhere.
It has previously been suggested that transposon silencing
may not be the sole function of mammalian pachytene
piRNAs, most of which are highly complementary only tothe locus from which they are transcribed and are derived
from unannotated intergenic regions depleted in transposon-derived sequences relative to the genome as a whole(Aravin et al. 2007). While their potential targets are thus unclear, piRNA clusters in rodents (Assis and Kondrashov2009) and humans (Lukic and Chen 2011) evolve rapidly under positive selection. Recent reports suggest that piRNAs
may target mRNAs to which they are only partially complementary, similar to miRNA:target interactions (Zhang et al.
2015). However, the biochemically confirmed functions of
PIWI proteins require extensive piRNA:target complementarity (Reuter et al. 2011). Finding that pachytene piRNAs
are made from sequences derived from an exogenous virus
suggests an alternative explanation for these observations,as well as a potential role for the transcription of piRNA precursors outside the germline: similar to CRISPR spacers(Mojica et al. 2005), they may serve an immunologic function
by targeting sequences foreign to the genome from which
they are derived (Sagy et al. 2014).
Genetic material laterally transferred from nonretroviral
viruses to host genomes has been noted for a wide varietyof pathogens and hosts (Zhdanov 1975; Belyi et al. 2010;
Gilbert and Feschotte 2010; Kapoor et al. 2010; Katzourakis
and Gifford 2010; Liu et al. 2010, 2011). Several of thesegene transfer events can be clearly attributed to retrotransposon activity (Ballinger et al. 2012), and in at least one casethe gene transfer is associated with viral resistance (Maori
et al. 2007). We hypothesize that, lacking RNA-dependent
RNA-polymerases used to amplify RNAs for viral silencing
in organisms for which this function is well-established(Baulcombe 2004; Rechavi et al. 2011), some metazoans
could use a multistep process: reverse transcription of viraltranscripts followed by RNA polymerization templated on
viral complementary DNA (cDNA). Sufficient reverse transcriptase activity acting on viral RNA is present in somemammalian cells to allow laboratory time-scale experiments(Pittoggi et al. 2006; Horie et al. 2010); LINE-1 ORF2p has
recently been suggested to be the responsible enzyme in human cells (Shimizu et al. 2014). While no function has been
ascribed to cDNA generated by this activity in mammals,www.rnajournal.org

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Parrish et al.

similar viral cDNA is a source of antiviral interfering RNA in
Drosophila (Goic et al. 2013).
The described interactions between infectious viruses
and the transposons that comprise the bulk of mammalianhost genomes are consistent with the hypothesis that transposons are symbionts integral to genomic stress responses(McClintock 1984), including antiviral immune responses(Zeng et al. 2014; Yu et al. 2015). The piRNA system inmammals is known to silence quasi-nonself transposon nucleic acids; our observations raise the hypothesis that, as for
CRISPR/Cas in prokaryotes, truly exogenous nonself nucleic
acids from infecting viruses can be targeted by piRNA-likeRNAs, but that this requires genetic information flow inan unexpected retrotransposon-dependent manner (Nuñez
et al. 2015). We refer to this hypothetical mechanism as
viral transcript reversion with anamnesic piRNA silencing(TRAPS) to indicate the host-directed nature of the reverse
transcription involved, its potential role in heritable immune
memory, and the specialized genomic loci involved in capturing the nonself information. Testing this hypothesis isof proximal relevance to human health, namely in arbovirus/vector interactions (Arensburger et al. 2011) and resistance to bornaviral disease.
MATERIALS AND METHODSEBLN annotation
The genomic locations of rodent and primate EBLNs have been described elsewhere (Belyi et al. 2010; Horie et al. 2010, 2013). Thesestudies defined EBLNs on the basis of potential ORFs or regions ofamino acid–based homologies with BDV N. In order to evaluate thehypothesis that small RNA generated from these loci could havebiological function, EBLN annotation in Table 2 is inclusive of theentire inserted BDV N mRNA-like sequence block (i.e., for primate
EBLNs, the sequence flanked by TSDs).

Small RNA sequencing
One 6-wk-old BALB/c mouse was purchased from Charles
River Laboratories, Japan. Testis total RNA including small RNA
fraction was collected from the mouse using a miRNeasy mini
Kit (QIAGEN). RNA quality was confirmed by 2100 Agilent
Bioanalyzer (Agilent Technologies). After quality confirmation,
cDNA libraries were constructed from the testis RNAs by TruSeq
small RNA sample prep kit (Illumina). Small RNA sequencing was
performed using an Illumina HiSeq (50SE) machine by Hokkaido
Systems Science. The sequence data were mapped onto mm9 using
BWA (Li and Durbin 2009) allowing up to two mismatches. Reads
mapping multiply were assigned at random to a single map site. Of
29,959,596 total reads, 27,968,611 reads were mapped, of which
3,184,031 mapped repetitively.

Mapping published small RNA-seq data
To analyze murine piRNAs corresponding to EBLNs, we used
GSM1096587 as small RNA data of 6-wk mouse testis (Li et al.
2013a). RNAs of 25 to 31 nt in length were mapped to mEBLNs

10

RNA, Vol. 21, No. 10

by Bowtie (Langmead et al. 2009) allowing up to two mismatches.
This bioinformatic analysis was performed using Galaxy (https://usegalaxy.org).

Cumulative binomial probability
The likelihood of x EBLNs or more, out of a total of n EBLNs fora given species, being found within a piRNA cluster if piRNA clusters occupy p percent of the genome was approximated as the probability P of x successes or more in n Bernoulli trials, each with aprobability p:
P(x) =n!
px (1 − p)n−x .
x!(n − x)!

piRNA cluster evolution
Ancestral piRNA clusters were as determined by Assis andKondrashov (2009), who considered rodent clusters in detail. To
identify the homologous primate clusters in human and marmoset,
LASTZ alignments of murine piRNA clusters to each of these species
were viewed using Ensembl and compared with annotations in Ha
et al. (2014) and Hirano et al. (2014).
Murine genes with 3′ UTRs giving rise to piRNA were determinedby Robine et al. (2009).

ACKNOWLEDGMENTS
We thank Masayuki Horie, Cedric Feschotte, Craig B. Wilen, Bryan
R. Cullen, Alexei A. Aravin, Andrew Z. Fire, Rafi Ahmed, Arnold
J. Levine, Sara Cherry, and P. Jeremy Wang for helpful discussions
and/or comments on the manuscript. N.F.P. thanks Bruce
R. Levin for encouraging a hunt for a mammalian CRISPR-like immune system. N.F.P. was supported by the Japan Society for the
Promotion of Science short-term postdoctoral fellowship award#PE13075. This study was supported in part by Funding Program
for Next Generation World-Leading Researchers (NEXT program),
KAKENHI grant numbers 26253027 and 26670225, and the Coreto-Core Program A, Advanced Research Networks from the Japan
Society for the Promotion of Science (JSPS) (K.T.); grants fromTakeda Science Foundation (K.T.); and KAKENHI grant numbers
25115508 and 25860336 from the Ministry of Education, Culture,
Science, Sports and Technology (MEXT) of Japan (T.H.).
Received April 16, 2015; accepted July 8, 2015.

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piRNAs derived from ancient viral processed pseudogenes astransgenerational sequence-specific immune memory in mammals
Nicholas F. Parrish, Kan Fujino, Yusuke Shiromoto, et al.
RNA published online August 17, 2015

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