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GENE SILENCING

An RNA exporter that enforces a no-export
policySmall RNAs guide nuclear Argonaute proteins to silence genomic target loci via recruitment of factors that lead toformation of repressive heterochromatin. Animal gonads use this pathway to repress transposable elements withPIWI-clade Argonaute proteins and their associated small RNAs called PIWI-interacting RNAs (piRNAs). Four
research groups now identify a protein complex that acts as a molecular bridge between the piRNA pathway andthe epigenetic silencing machinery.

David Homolka and Ramesh S. Pillai758mRNA export
CBCS

mall RNAs guide their associatednuclear Argonaute proteins togenomic loci for transcriptionalsilencing1–3. Drosophila melanogasterovaries express Piwi, a nuclear memberof the PIWI-clade Argonautes that is
responsible for transposon repression.
Guided to nascent transposon transcripts
by its associated piRNAs, Piwi recruits thecellular machinery that establishes histone
H3 lysine 9 trimethylation (H3K9me3),
a repressive chromatin mark, leading toco-transcriptional gene silencing. This
suppression of transposable elements
is essential to ensure genome integrity
and fertility. In this issue, Batki et al.4
report the identification of a protein
complex, termed SFiNX (for silencing
factor-interacting nuclear export variant),
which links the piRNA pathway to thegeneral heterochromatin machinery. It
is intriguing that this complex includes
a variant RNA exporter, nuclear export
factor 2 (Nxf2) that no longer functions inRNA export. Instead, it collaborates with
its interaction partner Panoramix (Panx)
to promote co-transcriptional silencing(Fig. 1). Together with related work from
Hannon5, Siomi6 and Yang7 laboratories,
this finding represents a substantial leap
in our knowledge of piRNA-mediatedco-transcriptional silencing.
Gene repression by Drosophila Piwi
acts through the deposition of H3K9me3
on target loci8–10. Histone modificationis mediated by members of the generalheterochromatin machinery, such as theH3K9me3 methyltransferase Eggless/SetDB1, the H3K9me2/3 reader protein HP1(Su(var)205) and the H3K4 demethylase
Lsd1 (Su(var)3-3) (refs. 11,12). However, howpiRNA-Piwi recognition of the nascent
RNA is molecularly linked to recruitmentof the heterochromatin machinery isCBCTHO

Nxf1 Nxt1

UAP56

H3K4me3

Translation

Pol II
Nxf1 Nxt1

Protein encoding locus

CBCTransposon silencing

SFiNXPiwi

Nxt1
Nxf2

Panoramix

H3K4me3
Pol IIHP1

Active transposable element

STOP

H3K9me3

Eggless

Eggless

HP1

Pol II
Silenced transposable element
Nucleus

Cytoplasm

Fig. 1 | Nxf2 promotes co-transcriptional silencing of transposon loci in the fly germ line. CanonicalRNA export (top) mediated by the export receptor Nxf1 and its co-factor Nxt1. The 5′ cap of the nascent
RNA that is still attached to the transcribing RNA polymerase II (Pol II) is bound by the cap-binding
complex (CBC). Together with subsequent splicing of the pre-mRNA, this leads to recruitment of thetranscription/export (TREX) complex composed of a multiprotein complex called THO and additional
factors including UAP56. In transposon silencing (bottom), targeting of nascent transposon transcripts
by the Piwi/piRNA complex results in recruitment of SFiNX. Within the SFiNX complex, the variant
RNA exporter Nxf2, which is defective in export, uses its RNA-binding domain to bind the nascentRNA, and Panoramix recruits the general heterochromatin machinery (Eggless and HP1). This results inaccumulation of silent H3K9me3 heterochromatin marks on transposon loci, shutting down transcription.

poorly understood. Classical genetics andwhole-genome screens using a transposon
repression assay13–15 identified Maelstrom8,
GTSF1 (ref. 16) and Panx11,12 as factors that are
required for transcriptional silencing by Piwi.
In a continuing quest for the recruitment

mechanism, the four research groups sought
to identify interaction partners of Panx.
This led to the discovery of Nxf2 (ref. 17), a
paralog of the general mRNA export factor
Nxf1 (ref. 18), and its co-factor Nxt1 (Fig. 1).
As previously shown for Panx, loss ofNature Structural & Molecular Biology | VOL 26 | SEPTEMBER 2019 | 758–765 | www.nature.com/nsmb

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Nxf2 leads to infertility and de-repression oftransposable elements in fly ovaries. This isaccompanied by increased RNA polymerase
II occupancy and a decrease in H3K9me3
marks on transposon loci.
The SFiNX complex (Panx-Nxf2-Nxt1)
has a direct role in silencing. Artificial
tethering of Nxf2 or Panx to the nascent
RNA of a reporter gene, or direct tethering
to DNA upstream of this reporter, shows
strong repression with a concomitant
increase of H3K9me3 marks. There is a clear
division of labor within the complex, which
the authors reveal by a combination of
domain mapping, crystal structure analyses
and tethering assays with interactioninterface mutants. Complex formation isobligate, as Panx and Nxf2 depend on eachother for protein stability. Furthermore,
nuclear accumulation of Nxf2 depends on
the nuclear localization signal of Panx. Thus,
it is a preformed complex that enters thenucleus. Using structural information from a
subcomplex comprising a fragment of Nxf2
(the UBA domain) and Panx (a C-terminalhelix), Batki et al.4 created mutants of Nxf2and Panx that no longer interact, but are
still stable. Individual tethering of thesemutants to a reporter RNA lead to notable
results. Only the interaction-deficient
Panx can function in target repression bydeposition of heterochromatin. In a related
study, Fabry et al.5 demonstrate that thisrepressive activity resides in the N-terminal
unstructured region of Panx. Although Panxalone could recruit the heterochromatin
machinery in the artificial tethering assay,
the interaction-deficient version wasinactive in rescue experiments, showing that
Panx requires Nxf2 to function in vivo.
Which role does Nxf2 play within thecomplex? The N-terminal part of Nxf2 hasa putative RNA-binding region consistingof an RNA recognition motif (RRM)
and a leucine-rich repeat (LRR), which
the authors demonstrate can bind RNA
in vitro. Nxf2 lacking this RNA-binding
domain is inactive in rescue experiments,
demonstrating a critical role for RNAbinding in vivo as well. Consistent with
an RNA-binding role in vivo, the domain
becomes dispensable for repression activity
when the deletion mutant is directly
tethered to a reporter RNA. Using RNA
cross-linking and immunoprecipitation(CLIP) experiments with tagged versions of
Nxf2, Murano et al.6 and Zhao et al.7 identify
transposon transcripts as in vivo substrates.
The authors also propose a role for Piwi indirecting access to such transcripts5. The
presence of Nxf2 within the SFiNX complex
might allow additional control over its
RNA-binding activity. In fact, crosslinking
mass spectrometry of the recombinant

complex reveals extensive intramolecularand intermolecular interactions with theRRM-LRR domain, indicating a potential‘closed’ state of the protein. It is likely thatinteraction with other factors and/or RNA
might convert this into an ‘open’ state toallow RNA binding4. Thus, Nxf2 has two
distinct functions: to bind the nascent
RNA from Piwi-targeted loci to stabilize
the complex, and to interact with Panx tomediate repression.
Co-transcriptional repression can beviewed as a dynamic hierarchical processultimately resulting in deposition of silent
histone marks. Murano et al.6 examined
Nxf2-tethered-reporter silencing in atime-resolved manner to arrive at theconclusion that silencing is a two-stepprocess. At early time points, reporter
silencing is achieved through reduced
RNA polymerase II occupancy, without
deposition of H3K9me3 marks6. Only
long-term silencing is correlated with thepresence of silent histone marks. Additional
experiments from Batki et al.4 show that theco-transcriptional silencing process requiresa hierarchical assembly of factors. Direct
tethering of heterochromatin factors like
Eggless, Lsd1 and HP1 to a reporter RNA
fails to induce repression, but tethering ofthese factors to the DNA upstream of thereporter leads to silencing. The authors
also find that tethering of Nxf2 to reporter
RNA induces stronger repression thantethering to DNA. The results point to avery specific hierarchical assembly of factors
when nascent RNA is used as an anchor forco-transcriptional repression.
The involvement of Nxf2 is interesting,as a previous study could not attribute
any export function to it17. The generalmRNA exporter Nxf1 transits throughthe nuclear pore complex interacting withthe phenylalanine-glycine (FG) repeat
nucleoporin meshwork within the porechannel. Examining crystal structures of theNxf2-Panx and Nxf2-Nxt1 fragments, Batkiet al.4 find that the two putative FG-repeat
binding pockets in Nxf2 are occluded
and might be unavailable for interaction
with FG nucleoporins, a fact Zhao et al.7
confirmed experimentally. Zhao et al.7 reportan additional mechanism by which Nxf2
might operate—a direct interaction between
Nxf2 and Nxf1 that prevents interaction ofNxf1 with FG-repeat nucleoporins7. Thus,
Nxf2 sabotages RNA export: it snatches
the nascent RNA away from Nxf1 to retain
it at the site of transcription and actively
allows co-transcriptional repression
via the SFiNX complex components.
Co-transcriptional repression of transposonloci offers a unique challenge to the RNA
export machinery. Under conditions of

Nature Structural & Molecular Biology | VOL 26 | SEPTEMBER 2019 | 758–765 | www.nature.com/nsmb

transposon derepression, for example,
in piRNA pathway mutants, the nascenttransposon RNA is a perfectly good cargo
for the general RNA export machinery.
However, when used as an anchor forco-transcriptional silencing, it should bedetained at the site of transcription as long aspossible. It is worth mentioning that studies
in fission yeast show that mutation of factors
involved in transcriptional elongation19
and general mRNA export20 favor small
RNA-mediated silencing, possibly because
these interventions increase retention of thenascent RNA at the site of transcription.
In conclusion, the four reports identify amolecular link between the nascent RNA andthe general silencing machinery. However,
events that lead from initial recognition of
nascent RNA by the piRNA-Piwi complex
to downstream recruitment of the SFiNXcomplex remain to be determined. The
molecular role of other factors implicatedin the silencing process also needs to beclarified. Finally, the existence of testisspecific Nxf paralogs in mammals, withsome being required for male fertility21,
offers the exciting possibility that similar
mechanisms might be operational in themammalian male germ line wherepiRNAs guide DNA methylation.
❐David Homolka and Ramesh S. Pillai*

Department of Molecular Biology, Science III,
University of Geneva, Geneva, Switzerland.
*e-mail: ramesh.pillai@unige.ch

Published online: 2 September 2019https://doi.org/10.1038/s41594-019-0294-y
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Competing interests

The authors declare no competing interests.

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