- Email: [email protected]
A CURIous Case of Molecular Kidnapping
Molecular Cell
Previews A CURIous Case of Molecular Kidnapping Dipayan Rudra1,* and Jonathan R. Warner2,* 1Academy
of Immunology and Microbiology, Institute for Basic Science (IBS), Pohang 37673, Republic of Korea of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA *Correspondence: [email protected] (J.R.W.), [email protected] (D.R.) http://dx.doi.org/10.1016/j.molcel.2016.10.036 2Department
In this issue of Molecular Cell, Albert et al. (2016) demonstrate how the production of rRNA and ribosomal proteins is coordinated through a two-step response to stress that requires cross-talk between a dedicated transcription factor and a ribosome assembly factor. To build a car, you need one brake pedal, two headlights, and four wheels for every engine. To build a ribosome, the yeast cell needs one of 79 ribosomal proteins (RPs) for each rRNA transcript. A rapidly growing cell must satisfy these needs with care, both to optimize its use of energy and to avoid potentially hazardous unbound RNA or protein molecules. In the case of S. cerevisiae, balance among the ribosomal proteins starts by having a dedicated transcription factor, Ifh1, which binds to a more general factor, Fhl1, to drive the transcription of the RP genes (Figure 1A). The presence of Ifh1 on the RP promoters is regulated by a complex series of kinases and their substrates that emanates from the key regulator of cell growth, TORC1 (Broach, 2012). TORC1 also promotes the transcription of rRNA, both by binding directly to rRNA genes and by protecting Rrn3, the key rRNA transcription factor, from ubiquitin-dependent degradation (Philippi et al., 2010). Just as the assembly of a car requires many tools, the assembly of a ribosome requires a team of more than 300 protein and RNA factors to effect the modification, trimming, and folding of the rRNA and its intimate melding with the RPs (Woolford and Baserga, 2013). Insight about how rRNA and RP gene transcription might be balanced came from the finding that Ifh1 is present in a complex (CURI) with Casein Kinase 2 (CK2) and two of the rRNA processing factors, Utp22 and Rrp7. This observation suggested that these assembly factors, freed from their job by a reduction in rRNA transcription, might sequester Ifh1 to reduce transcription of RP genes (Rudra et al., 2007). Now, Shore and colleagues (Albert et al., 2016) have confirmed and greatly expanded this
notion in several ways. They firmly establish that Utp22 is the key factor responsible for retaining Ifh1 in the CURI complex, and they show that it does so by interacting with specific sites within Ifh1 that are distinct from those necessary for its association with Fhl1 at the RP genes. Importantly, they demonstrate that the loss of Ifh1 from the RP genes requires both the reduction of RNA polymerase I (Pol I) transcription and the presence of Utp22. One of their key findings is that the repression of the RP genes in response to stress is a two-step process. The first step, occurring within 5 min, is the displacement of Ifh1 from the RP genes (Figure 1B). This depends on the inactivation of Sch9 and the subsequent nuclear localization of Stb3, which binds RP promoters to induce repressive chromatin modifications. Yet the authors suggest that a key intermediate remains to be identified. In the second step, the released Ifh1 binds Utp22 and is sequestered within the CURI complex to effect long-term repression of RP gene transcription (Figure 1C). The authors show that in this phase Ifh1 is relocated to the nucleolus, the home of Utp22, presumably until it is ‘‘ransomed’’ by improved growing conditions. In cells with reduced levels of Utp22, or in cells with constitutive rRNA transcription, Ifh1 returns to the RP genes within 20 min of rapamycin- or amino-acid-induced stress. Thus, sustained repression of RP genes requires Utp22 and perhaps other members of the CURI complex. Interestingly, overexpression of Utp22 has little effect on cell growth or on the presence of Ifh1 at RP genes unless Ifh1 has been mutated to remove potential sites of phosphorylation essential for its high affinity with Fhl1.
However, depletion of Utp22 leads to an increased level of Ifh1 at the RP promoters (Albert et al., 2016) as well as to increased transcription of RP genes (Rudra et al., 2007). Finally, Utp22 seems to be one of the few non-RP genes whose transcription is controlled by Ifh1, suggesting obvious feedback mechanisms. The observations of Albert et al. (2016) provide a useful opportunity to consider the affinities and the flux of these regulatory molecules. A recent analysis of the proteome of S. cerevisiae suggests that there is less than one Ifh1 molecule per RP gene, while there are around 3,000 copies each of Utp22 and Rrp7 (Kulak et al., 2014)—not surprising, since they must process nearly 2,500 ribosomes per minute. Yet the low abundance of Ifh1 means that its activity as a transcription factor may be exquisitely sensitive to the presence of Utp22 released from the rRNA transcription units. More intriguing yet is the yin-yang nature of the CURI complex: while Utp22 sequesters Ifh1, CK2 phosphorylates Ifh1 at the site necessary for strong binding to Fhl1 at the RP genes (Kim and Hahn, 2016), ensuring a fluid response to subtle alterations in growth conditions. The kinetics of the two-step process suggest that in the wild, where conditions in general do not change as acutely, this CURI-dependent molecular rheostat may be the major element that maintains balance between rRNA and the RPs. To what extent can we translate the results of Albert et al. (2016) to mammalian cells? The mRNAs for the many RPs are certainly coordinated, with high levels of each in tissues such as ovary and substantially lower levels in brain. Mammalian TORC1 is also known to play a role in the balance between rRNA and RPs. When
Molecular Cell 64, November 17, 2016 ª 2016 Elsevier Inc. 639
Molecular Cell
Previews A
B Exponential growth conditions
C Early phase of TORC1 inhibition
Late phase of TORC1 inhibition
Nucleolus
rRNA gene Pol I
Rrn3
Funconal SSU Processome
Rrn3
CK2 Utp22
Pol I
Pol I
Stalled SSU Processome
CK2 Rrp7
Utp22
Stalled SSU Processome
Rrn3 CK2
Rrp7
TORC1
Rrp7
I1 Utp22
Stb3/Rpd3/ others? I1
?
P Rap1
Fhl1
RPG
Rap1
Pol II
Rap1
Fhl1 Pol II
Rpd3 Fhl1 Stb3 Pol II
Nucleus
Figure 1. A Model Depicting the Early and Late Phases of the Repression of Ribosomal Protein Gene Transcription following Inactivation of TORC1 (A) During exponential growth, both the rDNA locus and ribosomal protein genes (RPG) are transcribed efficiently. The key RP transcription factor Ifh1 is associated with RP gene promoter via its interaction with the fork-head transcription factor Fhl1. Phosphorylation of Ifh1 strengthens this interaction. Nucleolar rRNA transcription and processing of the 35S rRNA continues normally. (B) Upon growth inhibition such as one mimicked by rapamycin treatment, TORC1 is inactivated, which leads to rapid release of Ifh1 from the RP gene promoter. While this phase is partially dependent on the nuclear entry of the transcription repressor Stb3 and its partner Rpd3, which are otherwise inactivated by the TORC1-dependent kinase Sch9 (not shown), the key events are yet to be determined. The rRNA processing components Utp22, Rrp7, and CK2 (sometimes called the UTP-C complex) associate with pre-rRNA only in the earliest stages of pre-rRNA processing, and they presumably become free shortly after Pol I transcription has ceased due to the inactivation of the TORC1-dependent Pol I activator Rrn3. The phosphorylation status of Ifh1 during this phase remains uncertain. (C) Ifh1 is stably sequestered in the CURI complex and physically located in the nucleolus in a manner that is entirely dependent on Utp22. Yet since CK2 can phosphorylate Ifh1 at the sites essential for strong binding to Fhl1 at the RP promoters, the CURI complex has the intriguing potential of both repressing and activating the transcription of RP genes.
activated in either normal or tumorigenic situations, TORC1 stimulates both transcription of rRNA and translation of RP mRNAs (reviewed in Gentilella et al., 2015). The importance of matching rRNA and RP production is evident from numerous human mutations that upset this balance—such as haploinsufficiency of a RP gene or a defect in rRNA processing—and cause serious pathologies termed ribosomopathies (reviewed in Danilova and Gazda, 2015). In some instances, the proximal cause of the pathology is the accumulation of p53 due to the sequestering of p53’s ubiquitin ligase, Hdm2, by the pre-ribosome complex of Rpl5, Rpl11, and 5S rRNA, which accumulates when there is imbalance between RP and rRNA production. One problem is that while Utp22, Rrp7, and CK2 all have
640 Molecular Cell 64, November 17, 2016
homologs in mammalian cells, Ifh1 does not. Indeed, no single transcription factor analogous to Ifh1 has been found for human RP genes. Could purification of a complex containing mammalian Utp22 lead to the identification of one? Another intriguing possibility is this: the recent observation that the mammalian RNA helicase DDX21 influences transcription at both rRNA and RP genes (Calo et al., 2015) suggests that mammalian cells may have evolved an entirely different mechanism to balance rRNA and RP production.
REFERENCES Albert, B., Knight, B., Merwin, J., Martin, V., Ottoz, D., Gloor, Y., Bruzzone, M.J., Rudner, A., and Shore, D. (2016). Mol. Cell 64, this issue, 720–733.
Broach, J.R. (2012). Genetics 192, 73–105. Calo, E., Flynn, R.A., Martin, L., Spitale, R.C., Chang, H.Y., and Wysocka, J. (2015). Nature 518, 249–253. Danilova, N., and Gazda, H.T. (2015). Dis. Model. Mech. 8, 1013–1026. Gentilella, A., Kozma, S.C., and Thomas, G. (2015). Biochim. Biophys. Acta 1849, 812–820. Kim, M.S., and Hahn, J.S. (2016). Biochim. Biophys. Acta 1859, 1004–1013. Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N., and Mann, M. (2014). Nat. Methods 11, 319–324. Philippi, A., Steinbauer, R., Reiter, A., Fath, S., Leger-Silvestre, I., Milkereit, P., Griesenbeck, J., and Tschochner, H. (2010). Nucleic Acids Res. 38, 5315–5326. Rudra, D., Mallick, J., Zhao, Y., and Warner, J.R. (2007). Mol. Cell. Biol. 27, 4815–4824. Woolford, J.L., Jr., and Baserga, S.J. (2013). Genetics 195, 643–681.
Previews A CURIous Case of Molecular Kidnapping Dipayan Rudra1,* and Jonathan R. Warner2,* 1Academy
of Immunology and Microbiology, Institute for Basic Science (IBS), Pohang 37673, Republic of Korea of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA *Correspondence: [email protected] (J.R.W.), [email protected] (D.R.) http://dx.doi.org/10.1016/j.molcel.2016.10.036 2Department
In this issue of Molecular Cell, Albert et al. (2016) demonstrate how the production of rRNA and ribosomal proteins is coordinated through a two-step response to stress that requires cross-talk between a dedicated transcription factor and a ribosome assembly factor. To build a car, you need one brake pedal, two headlights, and four wheels for every engine. To build a ribosome, the yeast cell needs one of 79 ribosomal proteins (RPs) for each rRNA transcript. A rapidly growing cell must satisfy these needs with care, both to optimize its use of energy and to avoid potentially hazardous unbound RNA or protein molecules. In the case of S. cerevisiae, balance among the ribosomal proteins starts by having a dedicated transcription factor, Ifh1, which binds to a more general factor, Fhl1, to drive the transcription of the RP genes (Figure 1A). The presence of Ifh1 on the RP promoters is regulated by a complex series of kinases and their substrates that emanates from the key regulator of cell growth, TORC1 (Broach, 2012). TORC1 also promotes the transcription of rRNA, both by binding directly to rRNA genes and by protecting Rrn3, the key rRNA transcription factor, from ubiquitin-dependent degradation (Philippi et al., 2010). Just as the assembly of a car requires many tools, the assembly of a ribosome requires a team of more than 300 protein and RNA factors to effect the modification, trimming, and folding of the rRNA and its intimate melding with the RPs (Woolford and Baserga, 2013). Insight about how rRNA and RP gene transcription might be balanced came from the finding that Ifh1 is present in a complex (CURI) with Casein Kinase 2 (CK2) and two of the rRNA processing factors, Utp22 and Rrp7. This observation suggested that these assembly factors, freed from their job by a reduction in rRNA transcription, might sequester Ifh1 to reduce transcription of RP genes (Rudra et al., 2007). Now, Shore and colleagues (Albert et al., 2016) have confirmed and greatly expanded this
notion in several ways. They firmly establish that Utp22 is the key factor responsible for retaining Ifh1 in the CURI complex, and they show that it does so by interacting with specific sites within Ifh1 that are distinct from those necessary for its association with Fhl1 at the RP genes. Importantly, they demonstrate that the loss of Ifh1 from the RP genes requires both the reduction of RNA polymerase I (Pol I) transcription and the presence of Utp22. One of their key findings is that the repression of the RP genes in response to stress is a two-step process. The first step, occurring within 5 min, is the displacement of Ifh1 from the RP genes (Figure 1B). This depends on the inactivation of Sch9 and the subsequent nuclear localization of Stb3, which binds RP promoters to induce repressive chromatin modifications. Yet the authors suggest that a key intermediate remains to be identified. In the second step, the released Ifh1 binds Utp22 and is sequestered within the CURI complex to effect long-term repression of RP gene transcription (Figure 1C). The authors show that in this phase Ifh1 is relocated to the nucleolus, the home of Utp22, presumably until it is ‘‘ransomed’’ by improved growing conditions. In cells with reduced levels of Utp22, or in cells with constitutive rRNA transcription, Ifh1 returns to the RP genes within 20 min of rapamycin- or amino-acid-induced stress. Thus, sustained repression of RP genes requires Utp22 and perhaps other members of the CURI complex. Interestingly, overexpression of Utp22 has little effect on cell growth or on the presence of Ifh1 at RP genes unless Ifh1 has been mutated to remove potential sites of phosphorylation essential for its high affinity with Fhl1.
However, depletion of Utp22 leads to an increased level of Ifh1 at the RP promoters (Albert et al., 2016) as well as to increased transcription of RP genes (Rudra et al., 2007). Finally, Utp22 seems to be one of the few non-RP genes whose transcription is controlled by Ifh1, suggesting obvious feedback mechanisms. The observations of Albert et al. (2016) provide a useful opportunity to consider the affinities and the flux of these regulatory molecules. A recent analysis of the proteome of S. cerevisiae suggests that there is less than one Ifh1 molecule per RP gene, while there are around 3,000 copies each of Utp22 and Rrp7 (Kulak et al., 2014)—not surprising, since they must process nearly 2,500 ribosomes per minute. Yet the low abundance of Ifh1 means that its activity as a transcription factor may be exquisitely sensitive to the presence of Utp22 released from the rRNA transcription units. More intriguing yet is the yin-yang nature of the CURI complex: while Utp22 sequesters Ifh1, CK2 phosphorylates Ifh1 at the site necessary for strong binding to Fhl1 at the RP genes (Kim and Hahn, 2016), ensuring a fluid response to subtle alterations in growth conditions. The kinetics of the two-step process suggest that in the wild, where conditions in general do not change as acutely, this CURI-dependent molecular rheostat may be the major element that maintains balance between rRNA and the RPs. To what extent can we translate the results of Albert et al. (2016) to mammalian cells? The mRNAs for the many RPs are certainly coordinated, with high levels of each in tissues such as ovary and substantially lower levels in brain. Mammalian TORC1 is also known to play a role in the balance between rRNA and RPs. When
Molecular Cell 64, November 17, 2016 ª 2016 Elsevier Inc. 639
Molecular Cell
Previews A
B Exponential growth conditions
C Early phase of TORC1 inhibition
Late phase of TORC1 inhibition
Nucleolus
rRNA gene Pol I
Rrn3
Funconal SSU Processome
Rrn3
CK2 Utp22
Pol I
Pol I
Stalled SSU Processome
CK2 Rrp7
Utp22
Stalled SSU Processome
Rrn3 CK2
Rrp7
TORC1
Rrp7
I1 Utp22
Stb3/Rpd3/ others? I1
?
P Rap1
Fhl1
RPG
Rap1
Pol II
Rap1
Fhl1 Pol II
Rpd3 Fhl1 Stb3 Pol II
Nucleus
Figure 1. A Model Depicting the Early and Late Phases of the Repression of Ribosomal Protein Gene Transcription following Inactivation of TORC1 (A) During exponential growth, both the rDNA locus and ribosomal protein genes (RPG) are transcribed efficiently. The key RP transcription factor Ifh1 is associated with RP gene promoter via its interaction with the fork-head transcription factor Fhl1. Phosphorylation of Ifh1 strengthens this interaction. Nucleolar rRNA transcription and processing of the 35S rRNA continues normally. (B) Upon growth inhibition such as one mimicked by rapamycin treatment, TORC1 is inactivated, which leads to rapid release of Ifh1 from the RP gene promoter. While this phase is partially dependent on the nuclear entry of the transcription repressor Stb3 and its partner Rpd3, which are otherwise inactivated by the TORC1-dependent kinase Sch9 (not shown), the key events are yet to be determined. The rRNA processing components Utp22, Rrp7, and CK2 (sometimes called the UTP-C complex) associate with pre-rRNA only in the earliest stages of pre-rRNA processing, and they presumably become free shortly after Pol I transcription has ceased due to the inactivation of the TORC1-dependent Pol I activator Rrn3. The phosphorylation status of Ifh1 during this phase remains uncertain. (C) Ifh1 is stably sequestered in the CURI complex and physically located in the nucleolus in a manner that is entirely dependent on Utp22. Yet since CK2 can phosphorylate Ifh1 at the sites essential for strong binding to Fhl1 at the RP promoters, the CURI complex has the intriguing potential of both repressing and activating the transcription of RP genes.
activated in either normal or tumorigenic situations, TORC1 stimulates both transcription of rRNA and translation of RP mRNAs (reviewed in Gentilella et al., 2015). The importance of matching rRNA and RP production is evident from numerous human mutations that upset this balance—such as haploinsufficiency of a RP gene or a defect in rRNA processing—and cause serious pathologies termed ribosomopathies (reviewed in Danilova and Gazda, 2015). In some instances, the proximal cause of the pathology is the accumulation of p53 due to the sequestering of p53’s ubiquitin ligase, Hdm2, by the pre-ribosome complex of Rpl5, Rpl11, and 5S rRNA, which accumulates when there is imbalance between RP and rRNA production. One problem is that while Utp22, Rrp7, and CK2 all have
640 Molecular Cell 64, November 17, 2016
homologs in mammalian cells, Ifh1 does not. Indeed, no single transcription factor analogous to Ifh1 has been found for human RP genes. Could purification of a complex containing mammalian Utp22 lead to the identification of one? Another intriguing possibility is this: the recent observation that the mammalian RNA helicase DDX21 influences transcription at both rRNA and RP genes (Calo et al., 2015) suggests that mammalian cells may have evolved an entirely different mechanism to balance rRNA and RP production.
REFERENCES Albert, B., Knight, B., Merwin, J., Martin, V., Ottoz, D., Gloor, Y., Bruzzone, M.J., Rudner, A., and Shore, D. (2016). Mol. Cell 64, this issue, 720–733.
Broach, J.R. (2012). Genetics 192, 73–105. Calo, E., Flynn, R.A., Martin, L., Spitale, R.C., Chang, H.Y., and Wysocka, J. (2015). Nature 518, 249–253. Danilova, N., and Gazda, H.T. (2015). Dis. Model. Mech. 8, 1013–1026. Gentilella, A., Kozma, S.C., and Thomas, G. (2015). Biochim. Biophys. Acta 1849, 812–820. Kim, M.S., and Hahn, J.S. (2016). Biochim. Biophys. Acta 1859, 1004–1013. Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N., and Mann, M. (2014). Nat. Methods 11, 319–324. Philippi, A., Steinbauer, R., Reiter, A., Fath, S., Leger-Silvestre, I., Milkereit, P., Griesenbeck, J., and Tschochner, H. (2010). Nucleic Acids Res. 38, 5315–5326. Rudra, D., Mallick, J., Zhao, Y., and Warner, J.R. (2007). Mol. Cell. Biol. 27, 4815–4824. Woolford, J.L., Jr., and Baserga, S.J. (2013). Genetics 195, 643–681.