RNAi is essential for pericentromeric heterochromatic formation in S. pombe, and although Dcr1, the initiator protein of this process, has been biochemically well described, its subcellular localization has remained elusive. In this issue of Developmental Cell, Emmerth et al. now show that Dcr1 is dynamically shuttling between nucleus and cytoplasm, adding new insight into the subcellular mechanics of RNAi.

Kaaij, L. J.

Ketting, R.F.

RNAi is essential for pericentromeric heterochromatic formation in S. pombe, and although Dcr1, the initiator protein of this process, has been biochemically well described, its subcellular localization has remained elusive. In this issue of Developmental Cell, Emmerth et al. now show that Dcr1 is dynamically shuttling between nucleus and cytoplasm, adding new insight into the subcellular mechanics of RNAi

Kaaij, Lucas J.T.

Ketting, René F.

Elsevier - Publisher Connector 

Dcr1 Tracked Down 

RNAi is essential for pericentromeric heterochromatic formation in S. pombe, and although Dcr1, the initiator protein of this process, has been biochemically well described, its subcellular localization has remained elusive. In this issue of Developmental Cell, Emmerth et al. now show that Dcr1 is dynamically shuttling between nucleus and cytoplasm, adding new insight into the subcellular mechanics of RNAi

Kaaij, L.J.

NARCIS 

Dcr1 tracked down

KNAW Repository

Developmental Cell
Previewstreatments of diseases that are impacted
by the loss of epigenetic maintenance.
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Pontoglio, M. (2009). Nat. Med.Dcr1 Tracked DownLucas J.T. Kaaij1 and Rene´ F. Ketting1,*
1Hubrecht Institute-KNAW & University Medical Centre Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
*Correspondence: r.ketting@hubrecht.eu
DOI 10.1016/j.devcel.2010.01.001
RNAi is essential for pericentromeric heterochromatic formation in S. pombe, and although Dcr1, the initiator
protein of this process, has been biochemically well described, its subcellular localization has remained
elusive. In this issue of Developmental Cell, Emmerth et al. now show that Dcr1 is dynamically shuttling
between nucleus and cytoplasm, adding new insight into the subcellular mechanics of RNAi.RNA interference (RNAi) is a mechanism
that uses small RNAs as specificity factors
to regulate gene expression. One of the
best-studied small RNA species is the
class of small interfering RNAs (siRNA).
siRNAs are processed from long dsRNA
molecules by dicer, an evolutionary well-
conserved RNaseIII-like ribonuclease,
containing additional functionalities like
a helicase domain and two RNA binding
domains: PAZ (Piwi Argonaute Zwille)
and dsRBD (double-strand RNA binding
domain). Processed siRNAs are loaded
into a cytoplasmic or a nuclear effector
complex called RNA-induced silencing
complex (RISC) or RNA induced transcrip-
tional silencing complex (RITS) respec-
tively. In these complexes, siRNAs are
bound by an Argonaute protein. Depend-
ing on the homology between siRNA and
target RNA and the type of Argonaute,
the target sequence can be silenced in
two different ways: through posttranscrip-
tional gene silencing (PTGS), which acts
directly on the target RNA itself by target
cleavage or translational inhibition, or
through chromatin dependent gene si-
lencing (CDGS), in which chromatin ofthe chromosomal locus producing the
homologous sequence is remodeled into
a repressive state (Carthew and Son-
theimer, 2009; Moazed, 2009).
An organism that has specialized its
RNAi machinery to a great extent to direct
chromatin modification is Schizosacchar-
omyces pombe (S. pombe). In S. pombe,
the formation of pericentromeric hetero-
chromatin is a layered process in which
first euchromatic histone modifications
are removed, and next, Clr4 methylates
H3K9 that is subsequently bound by
Swi6. Biochemical studies have identified
two distinct complexes that are essential
for this heterochromatin formation. The
Argonaute protein Ago1, the tryptophan
GW-motif-containing protein Tas3, and
the chromodomain protein Chp1 make up
the RITS complex, which physically inter-
acts with outer centromeric repeat tran-
scripts in an siRNA-dependent manner.
The RNA-directed RNA polymerase com-
plex (RDRC), which physically interacts
with RITS and amplifies the siRNA signal,
has three core components: Rdp1
(anRNA-directed RNA polymerase), Cid12
(a polyadenylation polymerase), and thepredicted helicase Hrr1. The interactions
among RITS, RDRC, and the centromeric
repeats are Dcr1 dependent, and Dcr1
itself has been reported to interact with
RDRC (Colmenares et al., 2007; Moazed,
2009; Motamedi et al., 2004; Verdel et al.,
2004). Previous reports based on Dcr1
overexpression studies reported counter-
intuitive cytoplasmic localization (Carmi-
chael et al., 2006). In this issue ofDevelop-
mental Cell, Emmerth and colleagues
readdress the question of the subcellular
Dcr1 localization (Emmerth et al., 2010).
First, live cell imaging was performed on
moderately expressed Dcr1-GFP fusion
protein, revealing a predominantly nuclear
localization. Interestingly, the nuclear
localization was not diffuse, but colocal-
ized with the nuclear pores in granule-like
structures on the inner side of the nuclear
membrane. These structures do not co-
localize with RITS components or with
chromatin, implying that siRNA generation
and target recognition could be physically
separated events. Interestingly in Arabi-
dopsis thaliana (A. thaliana) and in the
animal germline-specific Piwi pathway,
the processing of CDGS-associated
Dcr1 granule
siRNA processing?
Shuttling bottleneck?
RDRC
RITS
 Regulated Dcr1 shuttling
Nuclear pore
Cytoplasmic Dcr1 function?
Nascent transcript
Nuclear membrane
?
Pol II
siRNA processing?
Dcr1
Dcr1
dsRBDC33
Dcr1
ds
RB
D
dsRBDC33
C3
3
peri-centromeric Chromatin
Figure 1. Schematic of Dcr1 Cytoplasmic-Nuclear Shuttling
RITS binds via siRNA-mediated base-pairing to pericentromeric nascent transcripts and recruits RDRC.
Subsequently the nascent transcript is made double-stranded by the RDRC component Rdp1 and
processed into siRNAs by Dcr1. Emmerth et al. (2010) show that Dcr1 shuttles between nucleus and cyto-
plasm. They also show that the dsRBD domain of Dcr1 stimulates this trafficking behavior, with the C33
domain inhibiting nuclear export. This may explain the observed accumulation of Dcr1 at the inside of
the nuclear pores. Given the lack of trans RNAi activity and the fact that Rdp1 does not localize to pores,
Dcr1-mediated siRNA processing likely occurs in association with chromatin and not at the nuclear pores,
although this has not been addressed directly. It also remains unclear whether indeed RDRC-bound Dcr1
exchanges with nuclear pore-associated Dcr1.
Developmental Cell
Previewssmall RNAs also occurs in distinct,
granule-like nuclear structures, away
from the chromatin target (Klattenhoff
and Theurkauf, 2008; Li et al., 2006).
Although so far no experiments have
shown a clear mechanistic role for this
separation, an argument could be that it
would promote silencing in trans. How-
ever, in S. pombe, RNAi silencing appears
to occur mainly in cis, and thus physical
separation of processing and effector
complexes would only introduce prob-
lems (Bu¨hler et al., 2006). This, in com-
bination with the fact that even the
dsRNA-generating enzyme, Rdp1, does
not colocalize with the nuclear pore-asso-
ciated Dcr1 pool, suggests that the site of
Dcr1 activity is in association with RDRC,
and that the localization to the nuclear
pore serves some other function.
To investigate the mechanism behind
the observed Dcr1 localization, Emmerth
and colleagues (2010) introduced C-ter-
minal truncations in Dcr1, while retaining
catalytic activity. Upon the removal of 33
C-terminal amino acids (Dcr1D33) Dcr1
became cytoplasmic, resulting in loss of
pericentromeric siRNAs and silencing.
An obvious first thought would be that
this C33 domain of Dcr1 contains a
nuclear localization signal (NLS), but deli-
cate experiments show that this is not thecase. On the contrary, even adding an
NLS to Dcr1D33 under its endogenous
promoter does not restore nuclear locali-
zation. Furthermore, fluorescence loss in
photobleaching (FLIP) experiments on
cells expressing Dcr1D33 indicate that
Dcr1D33 shuttles between cytoplasm
and nucleus and that this is mediated by
the dsRBD domain of Dcr1 (Figure 1).
Interestingly, nuclear localization of a
larger C-terminal truncation of Dcr1
(Dcr1D103), also lacking the dsRBD
domain, is restored upon adding a canon-
ical NLS, indicating that the C33 domain
somehow specifically blocks dsRBD-
mediated nuclear export. Previous exper-
iments suggested that the interaction
between Dcr1 and Rdp1 required the
C-terminal 103 amino acids (Colmenares
et al., 2007), but from the data presented
by Emmerth et al. (2010), it is now clear
that this could be due to the cytoplasmic
mislocalization of Dcr1D103. It would
thus be interesting to see if nuclear local-
ized Dcr1D103 would still interact with
RDRC; the fact that nuclear Dcr1D103
rescues heterochromatin defects of Dcr1
mutant cells would suggest it does.
But why shuttle Dcr1 between cyto-
plasm and nucleus? And does the shut-
tling behavior relate to the Dcr1 granules
at the nuclear pores? Interestingly,Developmental CellEmmerth et al. (2010) provide data sug-
gesting that Dicer protein can be toxic to
cells, in a way that is independent of its
catalytic activity. Well-controlled Dicer
localization could be one of the mecha-
nisms to restrict unwanted Dicer activity,
and one way to achieve that would be to
impose a strong rate-limiting step at some
point in a transportation cycle (Figure 1).
The C33 domain of Dcr1 appears to do
just that: inhibiting nuclear export of
Dcr1 cycling between nucleus and cyto-
plasm, causing it to accumulate at the
nuclear pores, on the inside of the nucleus.
However, it should be noted that nuclear
Dcr1D103 assembles into granules much
like wild-type Dcr1, while still being toxic
to cells (Emmerth et al., 2010), indicating
that much more future work is required
to fully understand Dcr1 dynamics and
its potential regulation. Interestingly, of
the four A. thaliana DICER-like proteins,
the nuclear versions have an extended
C-terminal tail, just like Dcr1, indicating
that a similar localization mechanism
could be at play. Such a conservation of
Dicer shuttling behavior in plants and
perhaps also in animal systems would
have a big impact on our current thinking
about small RNA biogenesis and would
demand experiments aimed at unveiling
nuclear functions of Dicer in animal cells.
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