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Ribosomal 60S-subunit production: the final scene Célia Plisson-Chastang, Natacha Larburu & Pierre-Emmanuel Gleizes
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© 2015 Nature America, Inc. All rights reserved.
Newly synthesized 60S ribosomal subunits are licensed for translation through the release of the antiassociation factor eIF6. A new study shows by cryo–electron microscopy how eIF6 eviction results from a long-range allosteric cascade that involves SBDS, the protein mutated in Shwachman-Diamond syndrome. Formation of ribosomal subunits in eukaryotes is a complex multistep process that involves a large number of trans-acting factors in addition to the components of the mature subunits1,2. An increasing number of genetic diseases (ribosomopathies) and cancers are being linked to mutations affecting either these ribosome-assembly factors or ribosomal proteins3. Deciphering the mechanisms of eukaryotic ribosome synthesis represents a major goal toward understanding the functional effects of pathological mutations. The final steps of the 60S subunit–assembly pathway constitute a point of convergence of two distinct hematological disorders: Shwachman-Diamond syndrome (SDS), a congenital ribosomopathy that is linked to mutations in the protein SBDS and that frequently progresses to myelodysplastic syndromes and acute myeloid leukemia4; and T-cell acute lymphoblastic leukemia (T-ALL), which is associated with mutations in the ribosomal protein RPL10 (uL16)5. eIF6 joins pre-60S particles in the nucleolus and prevents their association with the 40S subunit by steric hindrance. Functional SBDS and RPL10 are both required for the release of eIF6 (Tif6 in yeast) from the late pre-60S particle in the cytoplasm4,6. SDSrelated mutations in SBDS have been shown to impair eIF6 release4, whereas RPL10 mutations in T-ALL affect a loop (peptidyl (P)-site loop) identified as a key component of the eIF6-release mechanism6,7. Genetic studies in budding yeast have also revealed the central role of the GTPase EFL1 in Tif6 eviction8–10, but the interplay of EFL1, RPL10 and SBDS in this mechanism has remained under debate. According to snapshots of this mechanism captured by high-resolution cryo-EM, Weis et al.11 now propose an explanation of how RPL10 and SBDS recruit the GTPase EFL1 to the pre-60S particle, which in turn evicts eIF6
through a large conformational rearrangement. This unifying model provides a direct explanation of how mutations in RPL10 or SBDS affect the release of eIF6. Weis et al.11 managed to isolate 60S particles containing eIF6 from the slime mold Dictyostelium discoideum and to reconstitute pre-60S-like complexes with recombinant human EFL1 and SBDS. Analysis of the cryo-EM images of this single preparation revealed the presence of three different complexes whose structures could be sorted and refined to a resolution ranging from 3–4 Å for the core of the pre-60S particle and to 5–9 Å for peripheral parts. This was sufficient to fit the NMR structures of SBDS10 and homology models of EFL1 and eIF6, as well as the 60Ssubunit components, into the electron density maps. The first complex displays SBDS bound to the eIF6-containing 60S particles. SBDS adopts an extended conformation, spanning the tRNA P site, the peptidyl transferase center, the entry of the ribosomal peptide-exit tunnel and the base of the stalk region. Importantly, the structure reveals interactions of SBDS with the RPL10 P-site loop, which is mutated in T-ALL. Consistently with this observation, introduction of these mutations in RPL10 in budding yeast decreased the recruitment to pre-60S particles of SDO1, the SBDS homolog in yeast, a result also observed in vitro7. The second complex imaged by cryo-EM corresponds to particles containing SBDS, EFL-1 and eIF6. Strikingly, while still positioned on the P site, SBDS undergoes a rotation that displaces it from the base of the stalk (‘open’ state), where it is replaced by EFL1. EFL1 makes extensive contacts with both SBDS and eIF6. In the third structure, EFL1 adopts an extended conformation on the surface of the pre-60S particles and binds to the sarcin-ricin loop, in a manner similar to that of the translational GTPases EF-G and EF-2. The contacts overlap with the binding sites of eIF6, which is absent from this particle. These three complexes clearly suggest a temporal sequence for eIF6 eviction, in which association of RPL10 with the pre-60S particle, a late event in the maturation pathway, allows recruitment of SDBS and EFL1 and thereby triggers an allosteric cascade leading
to the release of eIF6 several nanometers away (Fig. 1). Direct eviction of eIF6 by EFL1 has already been supported by the ability of EFL1 mutants favoring an extended conformation to promote eIF6 release in RPL10 P-site-loop mutants6. Strikingly, the structure of SBDS and its rotation in the presence of EFL1 are highly reminiscent of the structure of the bacterial ribosome-recycling factor RRF and its interaction with the elongation GTPase EF-G on the 50S subunit during translation termination12. EFL1 binding, accommodation and release may therefore be the rehearsal of the activity of the GTPases involved in translation, a ‘test drive’ for the 60S subunit7. Additional work is needed to clarify the precise regulation of GTP hydrolysis by EFL1 (ref. 10) as well as the temporal coordination of eIF6 release with respect to the other ribosome-assembly factors, such as NMD3, found in late pre-60S particles. These structures, coupled to functional analyses in yeast, reveal how several SDSrelated mutations impinge on SBDS function and eIF6 release. Whereas some of the mutated amino acids are involved in the initial interaction of SBDS with the P site of 28S rRNA, others are required for the rotation of SBDS to the open conformation or its stabilization through interaction with 28S rRNA. Similarly, mutations of the RPL10 P-site loop in T-ALL also impair eIF6 eviction by weakening SBDS binding to pre-60S particles7,11. As in other ribosomopathies, a constitutive defect in ribosome biogenesis could contribute to cancer progression by favoring the selection of mutations that perturb cell homeostasis13,14. Despite having a similar effect on 60S-subunit maturation, SBDS mutations promote acute myeloid leukemia, whereas RPL10 mutations promote acute lymphoid leukemia. This difference could stem from the genetic etiology of the two diseases, the former of which is inherited and the latter of which is sporadic. Intriguingly, congenital mutations in RPL10 have been linked to neurological disorders and autism15,16. The model proposed by Weis et al.11 offers a new structural framework to decipher the relative effects of RPL10 and SBDS mutations in these seemingly unrelated pathologies.
nature structural & molecular biology volume 22 number 11 November 2015
837
Célia Plisson-Chastang, Natacha Larburu and Pierre-Emmanuel Gleizes are at the Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France, and CNRS, UMR 5099, Toulouse, France. e-mail: [email protected]
news and views SBDS RPL10 mutations in T-ALL P-stalk
RPL10
GTP
RPL10
SBDS
EFL1
RPL10
RPL10
EFL1accomodated state
base P site
RPL10
Pi
SRL eIF6
GTP Peptideexit SBDS tunnel ‘closed’
SBDS mutations in SDS
eIF6
SBDS ‘open’
GTP GD
SBDS eIF6
P
eIF6 EFL1
Pre-60S particle
Translation-competent 60S subunit
This three-in-one structural study illuminates the final scene of the 60S-production process, of which additional intermediate sequences have already been captured, owing to recent advances in cryo-EM17–19. However, the complexity of ribosome biogenesis is highest at its early steps, thus making imaging of early complexes challenging. Other methodologies are therefore needed to dissect the molecular events associated with formation of these complexes. In that regard, Chaker-Margot et al.20 have proposed a new purification scheme to trap early preribosomal complexes on truncated pre-rRNAs. Using mass spectrometry, they describe several cotranscriptional assembly steps of the small-subunit processome, the maturation machinery that excises the 5′ external transcribed spacer in the pre-rRNA. This
new approach holds the promise of revealing more details of early steps in ribosome biogenesis, which may also shed further light on the ‘dark side’ of ribosomes in human diseases. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Henras, A.K. et al. Cell. Mol. Life Sci. 65, 2334–2359 (2008). 2. Thomson, E., Ferreira-Cerca, S. & Hurt, E. J. Cell Sci. 126, 4815–4821 (2013). 3. Danilova, N. & Gazda, H. Dis. Model. Mech. 8, 1013–1026 (2015). 4. Menne, T.F. et al. Nat. Genet. 39, 486–495 (2007). 5. De Keersmaecker, K. et al. Nat. Genet. 45, 186–190 (2013). 6. Bussiere, C., Hashem, Y., Arora, S., Frank, J. & Johnson, A.W. J. Cell Biol. 197, 747–759 (2012). 7. Sulima, S.O. et al. Nucleic Acids Res. 42, 2049–2063 (2014).
8. Senger, B. et al. Mol. Cell 8, 1363–1373 (2001). 9. Lo, K.Y. et al. Mol. Cell 39, 196–208 (2010). 10. Finch, A.J. et al. Genes Dev. 25, 917–929 (2011). 11. Weis, F. et al. Nat. Struct. Mol. Biol. 22, 914–919 (2015). 12. Yokoyama, T. et al. EMBO J. 31, 1836–1846 (2012). 13. Stumpf, C.R. & Ruggero, D. Curr. Opin. Genet. Dev. 21, 474–483 (2011). 14. Sulima, S.O. et al. Proc. Natl. Acad. Sci. USA 111, 5640–5645 (2014). 15. Klauck, S.M. et al. Mol. Psychiatry 11, 1073–1084 (2006). 16. Brooks, S.S. et al. Genetics 198, 723–733 (2014). 17. Bradatsch, B. et al. Nat. Struct. Mol. Biol. 19, 1234–1241 (2012). 18. Greber, B.J., Boehringer, D., Montellese, C. & Ban, N. Nat. Struct. Mol. Biol. 19, 1228–1233 (2012). 19. Leidig, C. et al. Nat Comun. 5, 3491 (2014). 20. Chaker-Margot, M., Hunziker, M., Barandun, J., Dill, B. & Klinge, S. Nat. Struct. Mol. Biol. 22, 920–923 (2015).
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© 2015 Nature America, Inc. All rights reserved.
Figure 1 Model of eIF6 release by SBDS and EFL1 from the pre-60S particle. Recruitment of SBDS in late cytoplasmic pre-60S particles requires binding to RPL10 (uL16) and 28S ribosomal RNA. EFL1–GTP binds to eIF6 and SBDS and favors SBDS rotation and repositioning (open conformation). EFL1 adopts an extended conformation overlapping eIF6-binding sites, thus leading to eIF6 eviction. Interaction of EFL1 with the sarcin-ricin loop (SRL) stimulates GTP hydrolysis and the release of EFL1 and SBDS. Mutations in RPL10 found in T-cell acute lymphoblastic leukemia (T-ALL) reduce binding of SBDS to the particle, as do mutations in SBDS found in Shwachman-Diamond syndrome (SDS). The mutations in both cases result in defective eIF6 eviction and impaired 60S-subunit production. The three central cartoons correspond to the structures determined by Weis et al.11.
838
volume 22 number 11 November 2015 nature structural & molecular biology
Ribosomal 60S-subunit production: the final scene Célia Plisson-Chastang, Natacha Larburu & Pierre-Emmanuel Gleizes
npg
© 2015 Nature America, Inc. All rights reserved.
Newly synthesized 60S ribosomal subunits are licensed for translation through the release of the antiassociation factor eIF6. A new study shows by cryo–electron microscopy how eIF6 eviction results from a long-range allosteric cascade that involves SBDS, the protein mutated in Shwachman-Diamond syndrome. Formation of ribosomal subunits in eukaryotes is a complex multistep process that involves a large number of trans-acting factors in addition to the components of the mature subunits1,2. An increasing number of genetic diseases (ribosomopathies) and cancers are being linked to mutations affecting either these ribosome-assembly factors or ribosomal proteins3. Deciphering the mechanisms of eukaryotic ribosome synthesis represents a major goal toward understanding the functional effects of pathological mutations. The final steps of the 60S subunit–assembly pathway constitute a point of convergence of two distinct hematological disorders: Shwachman-Diamond syndrome (SDS), a congenital ribosomopathy that is linked to mutations in the protein SBDS and that frequently progresses to myelodysplastic syndromes and acute myeloid leukemia4; and T-cell acute lymphoblastic leukemia (T-ALL), which is associated with mutations in the ribosomal protein RPL10 (uL16)5. eIF6 joins pre-60S particles in the nucleolus and prevents their association with the 40S subunit by steric hindrance. Functional SBDS and RPL10 are both required for the release of eIF6 (Tif6 in yeast) from the late pre-60S particle in the cytoplasm4,6. SDSrelated mutations in SBDS have been shown to impair eIF6 release4, whereas RPL10 mutations in T-ALL affect a loop (peptidyl (P)-site loop) identified as a key component of the eIF6-release mechanism6,7. Genetic studies in budding yeast have also revealed the central role of the GTPase EFL1 in Tif6 eviction8–10, but the interplay of EFL1, RPL10 and SBDS in this mechanism has remained under debate. According to snapshots of this mechanism captured by high-resolution cryo-EM, Weis et al.11 now propose an explanation of how RPL10 and SBDS recruit the GTPase EFL1 to the pre-60S particle, which in turn evicts eIF6
through a large conformational rearrangement. This unifying model provides a direct explanation of how mutations in RPL10 or SBDS affect the release of eIF6. Weis et al.11 managed to isolate 60S particles containing eIF6 from the slime mold Dictyostelium discoideum and to reconstitute pre-60S-like complexes with recombinant human EFL1 and SBDS. Analysis of the cryo-EM images of this single preparation revealed the presence of three different complexes whose structures could be sorted and refined to a resolution ranging from 3–4 Å for the core of the pre-60S particle and to 5–9 Å for peripheral parts. This was sufficient to fit the NMR structures of SBDS10 and homology models of EFL1 and eIF6, as well as the 60Ssubunit components, into the electron density maps. The first complex displays SBDS bound to the eIF6-containing 60S particles. SBDS adopts an extended conformation, spanning the tRNA P site, the peptidyl transferase center, the entry of the ribosomal peptide-exit tunnel and the base of the stalk region. Importantly, the structure reveals interactions of SBDS with the RPL10 P-site loop, which is mutated in T-ALL. Consistently with this observation, introduction of these mutations in RPL10 in budding yeast decreased the recruitment to pre-60S particles of SDO1, the SBDS homolog in yeast, a result also observed in vitro7. The second complex imaged by cryo-EM corresponds to particles containing SBDS, EFL-1 and eIF6. Strikingly, while still positioned on the P site, SBDS undergoes a rotation that displaces it from the base of the stalk (‘open’ state), where it is replaced by EFL1. EFL1 makes extensive contacts with both SBDS and eIF6. In the third structure, EFL1 adopts an extended conformation on the surface of the pre-60S particles and binds to the sarcin-ricin loop, in a manner similar to that of the translational GTPases EF-G and EF-2. The contacts overlap with the binding sites of eIF6, which is absent from this particle. These three complexes clearly suggest a temporal sequence for eIF6 eviction, in which association of RPL10 with the pre-60S particle, a late event in the maturation pathway, allows recruitment of SDBS and EFL1 and thereby triggers an allosteric cascade leading
to the release of eIF6 several nanometers away (Fig. 1). Direct eviction of eIF6 by EFL1 has already been supported by the ability of EFL1 mutants favoring an extended conformation to promote eIF6 release in RPL10 P-site-loop mutants6. Strikingly, the structure of SBDS and its rotation in the presence of EFL1 are highly reminiscent of the structure of the bacterial ribosome-recycling factor RRF and its interaction with the elongation GTPase EF-G on the 50S subunit during translation termination12. EFL1 binding, accommodation and release may therefore be the rehearsal of the activity of the GTPases involved in translation, a ‘test drive’ for the 60S subunit7. Additional work is needed to clarify the precise regulation of GTP hydrolysis by EFL1 (ref. 10) as well as the temporal coordination of eIF6 release with respect to the other ribosome-assembly factors, such as NMD3, found in late pre-60S particles. These structures, coupled to functional analyses in yeast, reveal how several SDSrelated mutations impinge on SBDS function and eIF6 release. Whereas some of the mutated amino acids are involved in the initial interaction of SBDS with the P site of 28S rRNA, others are required for the rotation of SBDS to the open conformation or its stabilization through interaction with 28S rRNA. Similarly, mutations of the RPL10 P-site loop in T-ALL also impair eIF6 eviction by weakening SBDS binding to pre-60S particles7,11. As in other ribosomopathies, a constitutive defect in ribosome biogenesis could contribute to cancer progression by favoring the selection of mutations that perturb cell homeostasis13,14. Despite having a similar effect on 60S-subunit maturation, SBDS mutations promote acute myeloid leukemia, whereas RPL10 mutations promote acute lymphoid leukemia. This difference could stem from the genetic etiology of the two diseases, the former of which is inherited and the latter of which is sporadic. Intriguingly, congenital mutations in RPL10 have been linked to neurological disorders and autism15,16. The model proposed by Weis et al.11 offers a new structural framework to decipher the relative effects of RPL10 and SBDS mutations in these seemingly unrelated pathologies.
nature structural & molecular biology volume 22 number 11 November 2015
837
Célia Plisson-Chastang, Natacha Larburu and Pierre-Emmanuel Gleizes are at the Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France, and CNRS, UMR 5099, Toulouse, France. e-mail: [email protected]
news and views SBDS RPL10 mutations in T-ALL P-stalk
RPL10
GTP
RPL10
SBDS
EFL1
RPL10
RPL10
EFL1accomodated state
base P site
RPL10
Pi
SRL eIF6
GTP Peptideexit SBDS tunnel ‘closed’
SBDS mutations in SDS
eIF6
SBDS ‘open’
GTP GD
SBDS eIF6
P
eIF6 EFL1
Pre-60S particle
Translation-competent 60S subunit
This three-in-one structural study illuminates the final scene of the 60S-production process, of which additional intermediate sequences have already been captured, owing to recent advances in cryo-EM17–19. However, the complexity of ribosome biogenesis is highest at its early steps, thus making imaging of early complexes challenging. Other methodologies are therefore needed to dissect the molecular events associated with formation of these complexes. In that regard, Chaker-Margot et al.20 have proposed a new purification scheme to trap early preribosomal complexes on truncated pre-rRNAs. Using mass spectrometry, they describe several cotranscriptional assembly steps of the small-subunit processome, the maturation machinery that excises the 5′ external transcribed spacer in the pre-rRNA. This
new approach holds the promise of revealing more details of early steps in ribosome biogenesis, which may also shed further light on the ‘dark side’ of ribosomes in human diseases. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Henras, A.K. et al. Cell. Mol. Life Sci. 65, 2334–2359 (2008). 2. Thomson, E., Ferreira-Cerca, S. & Hurt, E. J. Cell Sci. 126, 4815–4821 (2013). 3. Danilova, N. & Gazda, H. Dis. Model. Mech. 8, 1013–1026 (2015). 4. Menne, T.F. et al. Nat. Genet. 39, 486–495 (2007). 5. De Keersmaecker, K. et al. Nat. Genet. 45, 186–190 (2013). 6. Bussiere, C., Hashem, Y., Arora, S., Frank, J. & Johnson, A.W. J. Cell Biol. 197, 747–759 (2012). 7. Sulima, S.O. et al. Nucleic Acids Res. 42, 2049–2063 (2014).
8. Senger, B. et al. Mol. Cell 8, 1363–1373 (2001). 9. Lo, K.Y. et al. Mol. Cell 39, 196–208 (2010). 10. Finch, A.J. et al. Genes Dev. 25, 917–929 (2011). 11. Weis, F. et al. Nat. Struct. Mol. Biol. 22, 914–919 (2015). 12. Yokoyama, T. et al. EMBO J. 31, 1836–1846 (2012). 13. Stumpf, C.R. & Ruggero, D. Curr. Opin. Genet. Dev. 21, 474–483 (2011). 14. Sulima, S.O. et al. Proc. Natl. Acad. Sci. USA 111, 5640–5645 (2014). 15. Klauck, S.M. et al. Mol. Psychiatry 11, 1073–1084 (2006). 16. Brooks, S.S. et al. Genetics 198, 723–733 (2014). 17. Bradatsch, B. et al. Nat. Struct. Mol. Biol. 19, 1234–1241 (2012). 18. Greber, B.J., Boehringer, D., Montellese, C. & Ban, N. Nat. Struct. Mol. Biol. 19, 1228–1233 (2012). 19. Leidig, C. et al. Nat Comun. 5, 3491 (2014). 20. Chaker-Margot, M., Hunziker, M., Barandun, J., Dill, B. & Klinge, S. Nat. Struct. Mol. Biol. 22, 920–923 (2015).
npg
© 2015 Nature America, Inc. All rights reserved.
Figure 1 Model of eIF6 release by SBDS and EFL1 from the pre-60S particle. Recruitment of SBDS in late cytoplasmic pre-60S particles requires binding to RPL10 (uL16) and 28S ribosomal RNA. EFL1–GTP binds to eIF6 and SBDS and favors SBDS rotation and repositioning (open conformation). EFL1 adopts an extended conformation overlapping eIF6-binding sites, thus leading to eIF6 eviction. Interaction of EFL1 with the sarcin-ricin loop (SRL) stimulates GTP hydrolysis and the release of EFL1 and SBDS. Mutations in RPL10 found in T-cell acute lymphoblastic leukemia (T-ALL) reduce binding of SBDS to the particle, as do mutations in SBDS found in Shwachman-Diamond syndrome (SDS). The mutations in both cases result in defective eIF6 eviction and impaired 60S-subunit production. The three central cartoons correspond to the structures determined by Weis et al.11.
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volume 22 number 11 November 2015 nature structural & molecular biology
