Molecular Cell
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that SRPK1 can function as both an on-
cogene and a tumor suppressor depend-
ing on the context. The precise nature of
this context remains to be explored.
This work opens many questions as
to the nature of the mechanisms by
which SRPKs can mediate tumorigenesis
through regulation of alternative splicing
or by distinct functions, including acti-
vation of signaling pathways including
PI3K/Akt. It is notable that PHLPPs have
also been shown to modulate ERK
signaling (Li et al., 2014), whereas minimal
effects on ERK phosphorylation are
observed upon SRPK1 inactivation. Simi-
larly, the PHLPP1 and PHLPP2 isoforms
show specificity toward Akt1, Akt2, and
Akt3 (Brognard et al., 2007), but whether
this specificity is maintained in the context
of SRPK1 inactivation or overexpression
Fragile X Mental Retardation
Protein and the Ribosome
Yuriko Harigaya1,2 and Roy Parker1,2,*
1Howard Hughes Medical Institute
2Department of Chemistry and Biochemistry
University of Colorado, Boulder, CO 80303, USA
*Correspondence: roy.parker@colorado.edu
http://dx.doi.org/10.1016/j.molcel.2014.04.027
In this issue of Molecular Cell, Chen et al. (2014) provide evidence that FMRP represses translation by binding
the ribosome, suggesting a novel form of translational control.
The fragile X mental retardation protein
(FMRP) is an RNA-binding protein highly
expressed in brain, with functions in re-
pressing translation of specific mRNAs.
FMRP plays important roles in synaptic
plasticity and neurodevelopment (Pfeiffer
and Huber, 2009). Moreover, expansion
of a CGG repeat in the 50 end of the human
FMR1 gene can lead to DNA methylation,
loss of FMRP expression, and fragile X
syndrome (FXS), a form of intellectual
disability and autism (Pfeiffer and Huber,
2009). Strikingly, in mice and Drosophila,
behavioral and/or developmental defects
caused by FMRP overexpression or
loss of function can be suppressed or
330 Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc.
is not known. Regardless, this new study
reinforces the emerging concept that
molecules such as SRPK1 can function
as both oncogenes and tumor suppres-
sors in the context of inactivation and
overexpression. The challenge remains
to explore the specific mechanism that
accounts for both activities in human
tumors, which will be particularly impor-
tant for cancer therapeutics.
REFERENCES
Brognard, J., Sierecki, E., Gao, T., and Newton,
A.C. (2007). Mol. Cell 25, 917–931.
Cancer Genome Atlas Network (2012). Nature 490,
61–70.
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550–562.
Feng, G.S. (2012). Cancer Cell 21, 150–154.
enhanced by mutations in proteins con-
trolling translation (Sudhakaran et al.,
2014; Udagawa et al., 2013). This sug-
gests that understanding how FMRP
represses translation might lead to thera-
pies based on compensatory changes
in other components of translational
control. In this issue, Chen et al. (2014)
provide observations suggesting that the
Drosophila ortholog of FMRP (dFMRP),
and by implication human FMRP as well,
represses translation by directly binding
the ribosome.
To understand the mechanism of
dFMRP function, Chen et al. (2014) first
demonstrate that dFMRP inhibits transla-
Hayes, G.M., Carrigan, P.E., and Miller, L.J. (2007).
Cancer Res. 67, 2072–2080.
Li, X., Stevens, P.D., Liu, J., Yang, H., Wang, W.,
Wang, C., Zeng, Z., Schmidt, M.D., Yang, M.,
Lee, E.Y., and Gao, T. (2014). Gastroenterology
146, 1301–1312.e10.
Manning, B.D., and Cantley, L.C. (2007). Cell 129,
1261–1274.
Newton, A.C., and Trotman, L.C. (2014). Annu.
Rev. Pharmacol. Toxicol. 54, 537–558.
Sanidas, I., Polytarchou, C., Hatziapostolou, M.,
Ezell, S.A., Kottakis, F., Hu, L., Guo, A., Xie, J.,
Comb, M.J., Iliopoulos, D., and Tsichlis, P.N.
(2014). Mol. Cell 53, 577–590.
Wang, P., Zhou, Z., Hu, A., Ponte de Albuquerque,
C., Zhou, Y., Hong, L., Sierecki, E., Ajiro, M., Kruh-
lak, M., Harris, C., et al. (2014). Mol. Cell 54, this
issue, 378–391.
Zhou, Z., Qiu, J., Liu, W., Zhou, Y., Plocinik, R.M.,
Li, H., Hu, Q., Ghosh, G., Adams, J.A., Rosenfeld,
M.G., and Fu, X.D. (2012). Mol. Cell 47, 422–433.
tion in a cell-free system. Moreover, this
repression is reduced by either the
I244N mutation in one of two RNA-binding
KH domains in dFMRP or by deletion of
the dFMRP RGG domain, another RNA-
binding domain. The critical observation
is that dFMRP directly binds 80S ribo-
somes as assessed by gel filtration,
FRET-based assays, or protein crosslink-
ing. Importantly, dFMRP binding to the
ribosome is reduced by the I244N or
DRGG mutations, suggesting a functional
relationship between ribosome binding
and translation repression. Finally, the
authors solve a cryo-EM structure of
dFMRP bound to the ribosome wherein
Molecular Cell

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the KH domains of dFMRP specific classes of mRNAs

are positioned so they would by different mechanisms.
overlap with the P-site tRNA, Some aspects of this work
which suggests that by raise interesting mechanistic
binding the ribosome in questions. The structure of
this manner dFMRP might FMRP on the ribosome sug-
inhibit the process of transla- gests a steric clash with the
tion elongation due to steric P-site tRNA, which is not
conflicts. consistent with a block to
Additional work has sug- ongoing elongation where a
gested that the human peptidyl-tRNA would be in
FMRP blocks translation the P site. One possibility is
elongation in cells (Darnell that the structure of FMRP
et al., 2011). First, mapping on an elongating ribosome is
of FMRP-binding sites on different from the isolated
RNAs by crosslinking in vivo 80S subunits examined here.
showed that FMRP interacts Alternatively, FMRP might
primarily with the coding re- target ribosomes that are
gion of mRNAs. Second, not engaged in translation,
some FMRP remains associ- or promote translation termi-
ated with polysomes even nation in some manner and
after puromycin treatment, then remain bound to a com-
which releases elongating plex lacking a P-site tRNA
ribosomes. This observation (Figure 1).
suggests that FMRP is either Another interesting issue is
blocking puromycin action the precise role of the
on some ribosomes or is in a different FMRP domains in
Figure 1. A Convergent Model of FMRP Function
complex where the ribo- FMRP (red) binds certain primary/secondary RNA elements in mRNA (top two
ribosome binding and trans-
somes are not actually elon- panels) and then docks on 80S ribosome (blue) via the KH1 and KH2 domains lation repression. This is
gating. Interestingly, after pu- in a manner that causes steric conflicts between these domains and P-site important because although
tRNA (middle panel). This may lead to ribosome stall with or without pep-
romycin release mRNAs a mutation in the KH1 domain
tidyl-tRNA and eventually to formation of a complex consisting of mRNA and
predicted to be FMRP targets multiple stacked ribosomes (bottom panel), which is suggested by a previous and deletion of the RGG
are slightly deeper in the poly- study (Darnell et al., 2011). It is not known whether aminoacyl (A)- and exit domain had similar effects
some gradient that non- (E)-site tRNAs are present in the stacked ribosomes. on translation repression, the
FMRP targets, which is inter- RGG deletion had a much
preted to suggest FMRP is stronger effect on ribosome
responsible for increased ribosome reten- mRNA. In any case, once associated binding. This implies that additional inter-
tion on the mRNA. Consistent with this with a specific mRNA, FMRP is pro- actions are important for translation
model, the puromycin-resistant com- posed to interact with elongating ribo- repression, perhaps involving mRNA.
plexes containing FMRP appear to somes and interfere with their function. Further studies on the mechanism of
have several ribosomes complexed on This model makes several predictions translation repression, the role of different
an mRNP when examined by electron that could be tested in future work. For FMRP domains, and FMRP interactions
microscopy. example, FMRP addition to in vitro trans- with elongating ribosomes will be of
A convergent model of FMRP function lation systems should block translation interest.
can now be proposed with two salient after the formation of elongating 80S This work adds to the growing number
points (Figure 1). First, FMRP is pro- complexes and be most efficient on of proteins that interact with ribosomes
posed to interact with a specific subset mRNAs that contain FMRP-binding sites. and regulate translation elongation. Ex-
of mRNAs (Brown et al., 2001). The basis In addition, a re-examination of the exist- amples include but are not limited to an
of FMRP specificity is unclear and has ing FMRP-RNA crosslinks seen in vivo Ago-EF1A-PUM complex inhibiting trans-
been suggested to be due to ACUK or could determine if the FMRP-ribosome lation elongation on specific mRNAs
WGGA sequences, to G-quadruplexes, interaction seen in the cryo-EM is sup- (Friend et al., 2012), the Stm1 protein in
or to pseudoknots (reviewed in Chen ported by crosslinking to the rRNA yeast directly binding and inhibiting ribo-
et al., 2014). The interaction with the in vivo. somes in a manner that controls mRNA
ribosome might also complicate the However, additional research suggests decapping (Balagopal and Parker, 2011),
observed binding in vivo, as the distribu- that FMRP can also control translation by and eIF5A interacting with ribosomes at
tion of FMRP across the coding region inhibiting translation initiation on some proline codons to promote elongation
could be due to interactions with ribo- mRNAs in neurons (Napoli et al., 2008). (Gutierrez et al., 2013). These observa-
somes and not specific elements in the This suggests that FMRP might regulate tions suggest that, like transcription

Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc. 331


elongation, translation elongation may be
a key site of translational control, and
numerous regulators of translation could
function by targeting the elongating ribo-
some. Identifying such regulators, their
mechanism of action, and if/how they
target specific subclasses of mRNAs will
be of future interest.
REFERENCES
Balagopal, V., and Parker, R. (2011). RNA 17,
835–842.
Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Don-
nell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wil-
When Breaking Is Bad but Repair Is Worse
Brian S. Plosky1,*
1Molecular Cell, Cell Press, 600 Technology Square, 5th Floor, Cambridge, MA 02139, USA
*Correspondence: bplosky@cell.com
http://dx.doi.org/10.1016/j.molcel.2014.04.021
DNA double-strand breaks (DSBs) are a major source of genome instability; however, recent studies from
Lee et al. (2014) and Orthwein et al. (2014) show why, at least during mitosis, suppression of DSB repair is
important.
People can certainly be indecisive, but
what about our cells? As far as we can
tell, cells don’t really ‘‘think’’ about deci-
sions, yet the various checkpoints in the
cell cycle make it clear that they are reluc-
tant to commit. Cells respond to DNA
damage using checkpoints throughout
most of the cell cycle and can even
reverse mitotic entry up until late pro-
phase (Rieder and Cole, 1998). However,
once a vertebrate cell approaches a point
of no return in mitosis and commits to
breaking down the nuclear envelope, it
appears to realize that it has to take the
plunge. Mitotic cells seem to shut down
the response to DNA damage and pro-
ceed with mitosis even when there are
clearly unrepaired double-strand breaks
(DSBs) or even chromosome fragments
present (Rieder and Cole, 1998). While
there is little or no slowing of mitosis, no
evidence of repair, and no apparent
checkpoint, these breaks do not go unno-
ticed. DNA damage signaling in mitotic
332 Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc.
kinson, K.D., Keene, J.D., et al. (2001). Cell 107,
477–487.
Chen, E., Sharma, M.R., Shi, X., Agrawal, R.K.,
and Joseph, S. (2014). Mol. Cell 54, this issue,
407–417.
Darnell, J.C., Van Driesche, S.J., Zhang, C., Hung,
K.Y., Mele, A., Fraser, C.E., Stone, E.F., Chen, C.,
Fak, J.J., Chi, S.W., et al. (2011). Cell 146, 247–261.
Friend, K., Campbell, Z.T., Cooke, A., Kroll-Con-
ner, P., Wickens, M.P., and Kimble, J. (2012).
Nat. Struct. Mol. Biol. 19, 176–183.
Gutierrez, E., Shin, B.S., Woolstenhulme, C.J.,
Kim, J.R., Saini, P., Buskirk, A.R., and Dever, T.E.
(2013). Mol. Cell 51, 35–45.
cells acts as expected to the point where
MDC1 is recruited to phosphorylated his-
tone H2AX (Giunta et al., 2010). The next
step in DSB repair would typically involve
the recruitment of the E3 ligase RNF8,
which works with RNF168 to ubiquitinate
histone H2A and amplify the initial signal
from the DSB to recruit DNA repair pro-
teins such as BRCA1 and 53BP1. The bal-
ance between BRCA1 and 53BP1 recruit-
ment helps determine the choice between
homologous recombination (HR) and
nonhomologous end-joining (NHEJ). It is
at this step that the signal seems to get
cut off: in damaged mitotic cells, there
is no evidence that RNF8, 53BP1, or
BRCA1 associate with DSBs (Giunta
et al., 2010). Two recent papers, one in
Science from Orthwein et al. (2014) and
another in this issue of Molecular Cell
from Lee et al. (2014), give some clues
about how RNF8 and 53BP1 are excluded
from DSBs during mitosis. By uncovering
the mechanisms involved, they have been
Molecular Cell
Previews
Napoli, I., Mercaldo, V., Boyl, P.P., Eleuteri, B.,
Zalfa, F., De Rubeis, S., Di Marino, D., Mohr, E.,
Massimi, M., Falconi, M., et al. (2008). Cell 134,
1042–1054.
Pfeiffer, B.E., and Huber, K.M. (2009). Neuroscien-
tist 15, 549–567.
Sudhakaran, I.P., Hillebrand, J., Dervan, A., Das,
S., Holohan, E.E., Hülsmeier, J., Sarov, M.,
Parker, R., VijayRaghavan, K., and Ramaswami,
M. (2014). Proc. Natl. Acad. Sci. USA 111, E99–
E108.
Udagawa, T., Farny, N.G., Jakovcevski, M.,
Kaphzan, H., Alarcon, J.M., Anilkumar, S.,
Ivshina, M., Hurt, J.A., Nagaoka, K., Nalavadi,
V.C., et al. (2013). Nat. Med. 19, 1473–1477.
able to demonstrate why it is indeed
important to exclude these factors from
damaged chromatin during mitosis.
The two groups took different ap-
proaches to understand how DSB repair
is suppressed in mitosis. Orthwein et al.
(2014) found that Mdc1 is unable to bind
to RNF8 from mitotic extracts, but inhibit-
ing Cdk1 activity could restore the inter-
action. They identified T198 as a Cdk1
target on RNF8 and found that a nonphos-
phorylatable RNF8-T198A mutant was
recruited to mitotic DSBs. Examining a
step downstream of RNF8, they saw that
RNF8-T198A expression also allowed
recruitment of BRCA1, while it was not
sufficient for 53BP1 to localize to DSBs.
This suggested that there were at least
two separate mechanisms regulating
DSB repair in mitosis (Orthwein et al.,
2014).
Here, the story converges with the
efforts of Lee et al. (2014). They have
been interested in understanding how