Published online: November 13, 2015

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Misprocessed tRNA response targets
piRNA clusters
Soichiro Yamanaka & Haruhiko Siomi
Germline PIWI-interacting RNA (piRNA)
clusters have a characteristic transcriptional status; although they do not havetheir own promoters, both genomic strands
are transcribed, while splicing and 30
processing signals are neglected. How thistranscription is maintained remains
unknown. Molla-Herman et al (2015)
discovered that mutations in a tRNAprocessing factor cause the loss of transcription at some piRNA clusters, leading tosterility in Drosophila melanogaster. Thisdefect in piRNA cluster transcription isrestored by mutations in genes required forthe DNA damage checkpoint or replication.

See also: A Molla-Herman et al (December2015)

Growing cells are at risk of genome
instability during replication. One
source of this instability is replication fork blockage, because this can
induce collapse of the replisome, leading toa double-strand break (DSB) (Branzei &Foiani, 2010). A DSB is the most deleterious
form of DNA damage; however, to affectrepair, cells are equipped with the DNA
damage response pathway, which consistsof elaborate machinery and includes
proteins such as Chk2 (Ciccia & Elledge,2010). This pathway triggers the DNA repair
pathway and simultaneously arrests the cell
cycle through p53 activation. DSBs areoccasionally triggered by transcription–replication interference mediated by R-loop
formation (Helmrich et al, 2013). An R-loop
is a RNA–DNA hybrid structure enriched at
RNA polymerase-pausing sites. This structure causes genome instability, possiblyby blocking replication fork progression.
Interestingly, mutations in several RNAprocessing factors such as mRNA 30 -processing

factors cause the accumulation of R-loops
and concurrent DNA damage (Helmrichet al, 2013).
tRNA gene clusters are highly transcribedregions, making these sites collision hotspots between Pol III and the replication
machinery. This conflict causes a frequent
replication fork arrest, which can result in
DNA lesions, namely DNA fragile sites(Helmrich et al, 2013). However, it is notknown whether defects in tRNA processing
affect the genome stability.
During the tRNA maturation process, 50
leaders are removed from initial transcripts
by RNase P and 30 trailers are cleaved off byRNase Z (Jarrous & Gopalan, 2010). In thisissue of The EMBO Journal, Molla-Herman
and colleagues report that mutations inD. melanogaster Rpp30, a conserved subunitof RNase P, cause defects in oogenesis, leading to sterility (Molla-Herman et al, 2015).
Rpp30 mutations affect tRNA processing,resulting in the accumulation of tRNA transcripts with both 50 leader and 30 trailer
sequences. However, the same mutations do
not appear to affect the size and quantity of
most mature tRNAs. This suggests that theobserved defects in oogenesis are not caused
by a shortage of mature tRNAs but rather
result from the accumulation of misprocessed tRNAs. Mice that are mutant for thepre-tRNA splicing factor, CLP1, accumulate
misprocessed tRNA fragments, leading to a
progressive loss of motor neurons, whilecells are sensitized to p53 activation in
response to oxidative stress (Hanada et al,2013). These findings prompted MollaHerman et al (2015) to observe that thearrest of oogenesis in Rpp30 mutants ispartially rescued by inactivating p53,
although double mutant flies remain sterile.
In Drosophila, DNA damage activates ATM/Chk2 signaling, which in turn activates p53.

Fertility is restored in Rpp30; chk2 (a.k.a.,mnk) double-mutant flies, suggesting that
accumulation of misprocessed tRNAs in
Rpp30 mutants triggers the activation of
DNA damage checkpoints.
Interestingly, Molla-Herman et al (2015)
observed that PCNA, a key component of thereplication machinery, was mostly lost fromthe nucleus in Rpp30 mutants, suggesting acollapse of replication forks. Pol III also
formed aggregates in Rpp30 mutant nuclei,
which may also cause replication fork
collapse and replication stress. This wascorroborated with the finding that mutationsin claspin, a gene involved in the replicationstress checkpoint (Ciccia & Elledge, 2010),
partly rescue the oogenesis defect in Rpp30mutants. From these results, Molla-Hermanet al (2015) conclude that Rpp30 mutations
increase DNA replication stress in the ovary.
RNA-seq analysis then revealed asurprise. The number of piRNA cluster transcripts was decreased in Rpp30 mutants,
which coincided with cluster loci having
decreased levels of H3K9me modification, awell-characterized repressive histone mark,and de-repression of some transposableelements (TEs). piRNAs are processed in a
Dicer-independent manner from long(~150 Kb) precursor transcripts and protect
the genome in animal gonads by repressing
the threat of TE-mediated insertion mutagenesis (Iwasaki et al, 2015). They are transcribed from special genomic regions, calledpiRNA clusters, which contain a large
number of truncated TEs and are mostly
located in heterochromatic regions. In Drosophila, two major types of piRNA cluster
exist: some are transcribed uni-directionally(“uni-strand” clusters) and many others aretranscribed from both genomic strands(“dual-strand” clusters) (Iwasaki et al, 2015).
Transcription from uni-strand clusters

Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan. E-mail: awa403@keio.jp
DOI 10.15252/embj.201593322 | Published online 13 November 2015

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The EMBO Journal Vol 34 | No 24 | 2015ª 2015 The Authors

Published online: November 13, 2015

Soichiro Yamanaka & Haruhiko Siomi

The EMBO JournaltRNA cluster affects piRNA cluster

MaturepiRNAA
2 trans-effectpiRNA production
machinery

MisprocessedtRNApiRNA
precursor
1 cis-effect
DNA damage
responseet al (2015) provide clues as to how dualstrand piRNA clusters are transcribed andwill help to answer one of the mostoutstanding questions in the piRNA researchfield.

ReferencesBranzei D, Foiani M (2010) Maintaining genome
stability at the replication fork. Nat Rev Mol
Cell Biol 11: 208 – 219

piRNA cluster

Ciccia A, Elledge SJ (2010) The DNA damagetRNA
gene cluster

response: making it safe to play with knives.
MaturepiRNA

B
piRNA production
machinery

Mol Cell 40: 179 – 204
Hanada T, Weitzer S, Mair B, Bernreuther C,Wainger BJ, Ichida J, Hanada R, Orthofer M,Cronin SJ, Komnenovic V, Minis A, Sato F,piRNA
precursor

Mimata H, Yoshimura A, Tamir I, Rainer J, KoflerR, Yaron A, Eggan KC, Woolf CJ et al (2013) CLP1tRNA
gene cluster

Open chromatin
environment

H3K9
me3piRNA cluster

links tRNA metabolism to progressive motorneuron loss. Nature 495: 474 – 480
Helmrich A, Ballarino M, Nudler E, Tora L (2013)
Transcription-replication encounters,
consequences and genomic instability. NatStruct Mol Biol 20: 412 – 418
Ishizu H, Iwasaki YW, Hirakata S, Ozaki H, Iwasaki
W, Siomi H, Siomi MC (2015) Somatic primary

Figure 1. How tRNA misprocessing affects piRNA clusters.
(A) cis-effect (1): In Rpp30 mutants, the DNA damage response triggered by replication fork collapse inhibits
piRNA cluster transcription. trans-effect (2): In Rpp30 mutants, misprocessed tRNAs themselves inactivate thepiRNA production machinery, leading to piRNA loss. (B) In wild type, transcription at tRNA genes confers achromatin environment suitable for piRNA transcription.

piRNA biogenesis driven by cis-acting RNA
elements and trans-acting Yb. Cell Rep 12:
429 – 440
Iwasaki YW, Siomi MC, Siomi H (2015) PIWIinteracting RNA: its biogenesis and functions.
Annu Rev Biochem 84: 405 – 433

including flamenco locus is initiated fromdiscrete promoter regions, which gives rise
to long precursor transcripts with shortmotifs that recruit Yb, a core component of
the piRNA-processing machinery (Ishizuet al, 2015). In contrast, dual-strand clusters
do not have their own promoters but instead
appear to be transcribed as read-throughproducts from flanking genes (Mohn et al,2014). While it is counterintuitive, dualstrand cluster transcription requires loci tobe H3K9me modified, which is recognized
by a germline-specific HP1 family protein,
Rhino (Klattenhoff et al, 2009).
Molla-Herman et al (2015) also foundthat the levels of piRNAs derived fromdual-strand clusters were dramaticallydecreased in Rpp30 mutants, while unistrand piRNAs were produced normally.
piRNA production from dual-strand clusters
was restored in Rpp30; chk2 doublemutants. Intriguingly, dual-strand clusters,
but not uni-strand clusters, often reside neartRNA gene clusters. This correlation led
Molla-Herman et al (2015) to propose that
replication stress at tRNA gene loci mayª 2015 The Authorscause a cis-effect on nearby dual-strandpiRNA clusters to arrest their transcription,
probably by affecting chromatin structure(Fig 1A). Wild-type tRNA gene clusters may,therefore, promote a permissive transcription conformation for dual-strand piRNA
clusters located in heterochromatin (Fig 1B)(Yamanaka et al, 2014). Supporting thismodel, tRNA gene loci have been implicatedin various large-scale gene regulatory
processes, including insulator activity andestablishment of chromatin boundaries(Noma et al, 2006). In this regard, it will beinteresting to see whether the expression ofgenes located near tRNA gene loci is affectedin Clp1-mutant mice, particularly in theirbrains where misprocessed tRNA fragments
accumulate (Hanada et al, 2013). However,the possibility remains that the misprocessed
tRNAs may act in trans to specifically affectfactors required for dual-strand cluster transcription, such as Rhino (Fig 1A). In thiscase, it is difficult to explain how piRNA
production from dual-strand clusters is
restored in Rpp30; chk2 double mutants. For
either scenario, the findings of Molla-Herman

Jarrous N, Gopalan V (2010) Archaeal/eukaryal
RNase P: subunits, functions and RNA
diversification. Nucleic Acids Res 38:
7885 – 7894
Klattenhoff C, Xi H, Li C, Lee S, Xu J, Khurana JS,Zhang F, Schultz N, Koppetsch BS, Nowosielska
A, Seitz H, Zamore PD, Weng Z, Theurkauf WE(2009) The Drosophila HP1 homolog Rhino isrequired for transposon silencing and piRNA
production by dual-strand clusters. Cell 138:
1137 – 1149
Mohn F, Sienski G, Handler D, Brennecke J (2014)
The rhino-deadlock-cutoff complex licenses
noncanonical transcription of dual-strand piRNA
clusters in Drosophila. Cell 157: 1364 – 1379
Molla-Herman A, Vallés AM, Ganem-Elbaz C,
Antoniewski C, Huynh JR (2015) tRNAprocessing defects induce replication stress andChk2-dependent disruption of piRNAtranscription. EMBO J 34: 3009 – 3027
Noma K, Cam HP, Maraia RJ, Grewal SI (2006) A rolefor TFIIIC transcription factor complex ingenome organization. Cell 125: 859 – 872
Yamanaka S, Siomi MC, Siomi H (2014) piRNAclusters and open chromatin structure. Mobile
DNA 5: 22

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