- Email: [email protected]
Fragile X Mental Retardation Protein and the Ribosome
Molecular Cell
Previews that SRPK1 can function as both an oncogene and a tumor suppressor depending 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 activation 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. Similarly, 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
is not known. Regardless, this new study reinforces the emerging concept that molecules such as SRPK1 can function as both oncogenes and tumor suppressors 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 important 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. Engelman, J.A. (2009). Nat. Rev. Cancer 9, 550–562. Feng, G.S. (2012). Cancer Cell 21, 150–154.
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., Kruhlak, 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.
Fragile X Mental Retardation Protein and the Ribosome Yuriko Harigaya1,2 and Roy Parker1,2,* 1Howard
Hughes Medical Institute of Chemistry and Biochemistry University of Colorado, Boulder, CO 80303, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.04.027 2Department
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 repressing 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
enhanced by mutations in proteins controlling translation (Sudhakaran et al., 2014; Udagawa et al., 2013). This suggests that understanding how FMRP represses translation might lead to therapies 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-
330 Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc.
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 RNAbinding domain. The critical observation is that dFMRP directly binds 80S ribosomes as assessed by gel filtration, FRET-based assays, or protein crosslinking. 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
Previews 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 suginhibit the process of translagests 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 sugongoing 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 retarget ribosomes that are gion of mRNAs. Second, not engaged in translation, some FMRP remains associor promote translation termiated with polysomes even nation in some manner and after puromycin treatment, then remain bound to a comwhich 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 riboribosome binding and transFMRP (red) binds certain primary/secondary RNA elements in mRNA (top two somes are not actually elonlation repression. This is panels) and then docks on 80S ribosome (blue) via the KH1 and KH2 domains in a manner that causes steric conflicts between these domains and P-site gating. Interestingly, after puimportant because although tRNA (middle panel). This may lead to ribosome stall with or without pepromycin 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 and deletion of the RGG multiple stacked ribosomes (bottom panel), which is suggested by a previous study (Darnell et al., 2011). It is not known whether aminoacyl (A)- and exit are slightly deeper in the polydomain had similar effects (E)-site tRNAs are present in the stacked ribosomes. some gradient that nonon translation repression, the FMRP targets, which is interRGG 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 intertion 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 example, FMRP addition to in vitro trans- with elongating ribosomes will be of microscopy. 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. Exof 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 transbeen 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 ribosomes in a manner that controls mRNA et al., 2014). The interaction with the in vivo. 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 observasomes 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
Molecular Cell
Previews elongation, translation elongation may be a key site of translational control, and numerous regulators of translation could function by targeting the elongating ribosome. Identifying such regulators, their mechanism of action, and if/how they target specific subclasses of mRNAs will be of future interest.
kinson, K.D., Keene, J.D., et al. (2001). Cell 107, 477–487.
REFERENCES
Friend, K., Campbell, Z.T., Cooke, A., Kroll-Conner, P., Wickens, M.P., and Kimble, J. (2012). Nat. Struct. Mol. Biol. 19, 176–183.
Balagopal, V., and Parker, R. (2011). RNA 17, 835–842. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wil-
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.
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.
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). Neuroscientist 15, 549–567. Sudhakaran, I.P., Hillebrand, J., Dervan, A., Das, S., Holohan, E.E., Hu¨lsmeier, 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.
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: [email protected] 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 decisions, yet the various checkpoints in the cell cycle make it clear that they are reluctant to commit. Cells respond to DNA damage using checkpoints throughout most of the cell cycle and can even reverse mitotic entry up until late prophase (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 proceed 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 unnoticed. DNA damage signaling in mitotic
cells acts as expected to the point where MDC1 is recruited to phosphorylated histone 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 proteins such as BRCA1 and 53BP1. The balance between BRCA1 and 53BP1 recruitment 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
332 Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc.
able to demonstrate why it is indeed important to exclude these factors from damaged chromatin during mitosis. The two groups took different approaches 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 inhibiting Cdk1 activity could restore the interaction. They identified T198 as a Cdk1 target on RNF8 and found that a nonphosphorylatable 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
Previews that SRPK1 can function as both an oncogene and a tumor suppressor depending 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 activation 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. Similarly, 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
is not known. Regardless, this new study reinforces the emerging concept that molecules such as SRPK1 can function as both oncogenes and tumor suppressors 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 important 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. Engelman, J.A. (2009). Nat. Rev. Cancer 9, 550–562. Feng, G.S. (2012). Cancer Cell 21, 150–154.
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., Kruhlak, 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.
Fragile X Mental Retardation Protein and the Ribosome Yuriko Harigaya1,2 and Roy Parker1,2,* 1Howard
Hughes Medical Institute of Chemistry and Biochemistry University of Colorado, Boulder, CO 80303, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.04.027 2Department
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 repressing 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
enhanced by mutations in proteins controlling translation (Sudhakaran et al., 2014; Udagawa et al., 2013). This suggests that understanding how FMRP represses translation might lead to therapies 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-
330 Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc.
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 RNAbinding domain. The critical observation is that dFMRP directly binds 80S ribosomes as assessed by gel filtration, FRET-based assays, or protein crosslinking. 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
Previews 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 suginhibit the process of translagests 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 sugongoing 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 retarget ribosomes that are gion of mRNAs. Second, not engaged in translation, some FMRP remains associor promote translation termiated with polysomes even nation in some manner and after puromycin treatment, then remain bound to a comwhich 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 riboribosome binding and transFMRP (red) binds certain primary/secondary RNA elements in mRNA (top two somes are not actually elonlation repression. This is panels) and then docks on 80S ribosome (blue) via the KH1 and KH2 domains in a manner that causes steric conflicts between these domains and P-site gating. Interestingly, after puimportant because although tRNA (middle panel). This may lead to ribosome stall with or without pepromycin 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 and deletion of the RGG multiple stacked ribosomes (bottom panel), which is suggested by a previous study (Darnell et al., 2011). It is not known whether aminoacyl (A)- and exit are slightly deeper in the polydomain had similar effects (E)-site tRNAs are present in the stacked ribosomes. some gradient that nonon translation repression, the FMRP targets, which is interRGG 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 intertion 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 example, FMRP addition to in vitro trans- with elongating ribosomes will be of microscopy. 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. Exof 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 transbeen 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 ribosomes in a manner that controls mRNA et al., 2014). The interaction with the in vivo. 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 observasomes 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
Molecular Cell
Previews elongation, translation elongation may be a key site of translational control, and numerous regulators of translation could function by targeting the elongating ribosome. Identifying such regulators, their mechanism of action, and if/how they target specific subclasses of mRNAs will be of future interest.
kinson, K.D., Keene, J.D., et al. (2001). Cell 107, 477–487.
REFERENCES
Friend, K., Campbell, Z.T., Cooke, A., Kroll-Conner, P., Wickens, M.P., and Kimble, J. (2012). Nat. Struct. Mol. Biol. 19, 176–183.
Balagopal, V., and Parker, R. (2011). RNA 17, 835–842. Brown, V., Jin, P., Ceman, S., Darnell, J.C., O’Donnell, W.T., Tenenbaum, S.A., Jin, X., Feng, Y., Wil-
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.
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.
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). Neuroscientist 15, 549–567. Sudhakaran, I.P., Hillebrand, J., Dervan, A., Das, S., Holohan, E.E., Hu¨lsmeier, 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.
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: [email protected] 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 decisions, yet the various checkpoints in the cell cycle make it clear that they are reluctant to commit. Cells respond to DNA damage using checkpoints throughout most of the cell cycle and can even reverse mitotic entry up until late prophase (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 proceed 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 unnoticed. DNA damage signaling in mitotic
cells acts as expected to the point where MDC1 is recruited to phosphorylated histone 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 proteins such as BRCA1 and 53BP1. The balance between BRCA1 and 53BP1 recruitment 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
332 Molecular Cell 54, May 8, 2014 ª2014 Elsevier Inc.
able to demonstrate why it is indeed important to exclude these factors from damaged chromatin during mitosis. The two groups took different approaches 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 inhibiting Cdk1 activity could restore the interaction. They identified T198 as a Cdk1 target on RNF8 and found that a nonphosphorylatable 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