news and views substrate-binding site, as occurs in the rockerswitch mechanism. On this 50th anniversary of Jardetzky’s alternating-access proposal, a wealth of available structural and biophysical information on many transport proteins is allowing researchers to visualize the physical reality of his theoretical proposal. Subtle variations in the complex process of membrane transport can now begin to be teased apart, and the alternating-access model can be refined to include translation and rotation of the protein—two properties that Jardetzky excluded from the ‘allosteric pump’ model1. In summary, these studies have provided two new examples of transporters that use an elevator mechanism. It is now clear
that this mechanism is not unique to the glutamate transporter family, and there may be many more transport proteins—unrelated in sequence and varied in biological function— that also use a twisting elevator to move their substrates across membranes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Jardetzky, O. Nature 211, 969–970 (1966). 2. Yan, N. Annu. Rev. Biophys. 44, 257–283 (2015). 3. Reyes, N., Ginter, C. & Boudker, O. Nature 462, 880–885 (2009). 4. Mulligan, C. et al. Nat. Struct. Mol. Biol. 23, 256–263 (2016). 5. Coincon, M. et al. Nat. Struct. Mol. Biol. 23, 248–255 (2016).
6. Mancusso, R., Gregorio, G.G., Liu, Q. & Wang, D.N. Nature 491, 622–626 (2012). 7. Boudker, O., Ryan, R.M., Yernool, D., Shimamoto, K. & Gouaux, E. Nature 445, 387–393 (2007). 8. Crisman, T.J., Qu, S., Kanner, B.I. & Forrest, L.R. Proc. Natl. Acad. Sci. USA 106, 20752–20757 (2009). 9. Vergara-Jaque, A., Fenollar-Ferrer, C., Kaufmann, D. & Forrest, L.R. Front. Pharmacol. 6, 183 (2015). 10. Hunte, C. et al. Nature 435, 1197–1202 (2005). 11. Lee, C. et al. Nature 501, 573–577 (2013). 12. Lee, C. et al. J. Gen. Physiol. 144, 529–544 (2014). 13. Paulino, C., Wöhlert, D., Kapotova, E., Yildiz, Ö. & Kühlbrandt, W. eLife 3, e03583 (2014). 14. Bolla, J.R. et al. Nat. Commun. 6, 6874 (2015). 15. Su, C.C. et al. Cell Rep. 11, 61–70 (2015). 16. Hu, N.J., Iwata, S., Cameron, A.D. & Drew, D. Nature 478, 408–411 (2011). 17. Zhou, X. et al. Nature 505, 569–573 (2014). 18. Wohlert, D., Grotzinger, M.J., Kuhlbrandt, W. & Yildiz, O. eLife 4, (2015). 19. Slotboom, D.J. Nat. Rev. Microbiol. 12, 79–87 (2014).
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© 2016 Nature America, Inc. All rights reserved.
A new communication hub in the RNA world Megan Mayerle & Christine Guthrie During assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), the RNA-binding protein (RBP) Gemin5 recognizes the snRNP code and interacts with the large Gemin2–SMN complex. So et al. now find that Gemin2 also interacts with U1-70K, thereby conferring a preferential advantage on U1 snRNP assembly, and they extrapolate that SMN–Gemin2 serves a general ribonucleoprotein-exchange function.
The U1 snRNP has been best characterized regarding its role in precursor (pre)-mRNA splicing; however, its abundance in cells far exceeds that necessary for splicing regulation1. Furthermore, U1 is the only snRNP that localizes to intronless genes2, and it has been implicated in the regulation of transcription initiation as well as pre-mRNA length via its interactions with the polyadenylation machinery3,4. However, the mechanism through which cells maintain high levels of U1 relative to the other components of the spliceosome is not well understood. In this issue, So et al.5 reveal that small nuclear RNAs (snRNAs) compete for access to SMN, a protein complex required for snRNP maturation. They show that the unrelated RNA-binding protein (RBP) U1-70K gives U1 an advantage in this competition for SMN access, thus explaining the cellular overabundance of U1. Furthermore, these results hint that, rather than simply being a spliceosome-assembly factor, SMN may act as a hub for ribonucleoprotein (RNP) exchange, where a diverse set of RNPs swap their cargo Megan Mayerle and Christine Guthrie are at the Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. e-mail: [email protected]
RNAs (Fig. 1). These findings may also explain the widespread perturbations in RNA metabolism observed in the neurological disease spinal muscle atrophy (SMA). So et al.5 have established that U1 snRNA has two potential pathways to interact with SMN: (i) the canonical pathway used by all snRNAs, which requires the RBP Gemin5, and (ii) a unique pathway that makes use of U1-70K. Through a series of pulldown assays designed to assess the roles of U1 stem-loop 1 and the snRNP code in assembly of the Sm core, the authors have demonstrated that depletion of Gemin5 abolishes U4 snRNA’s ability to interact with the SMN complex, as expected. In contrast, the interaction between U1 snRNA and SMN is only modestly affected by depletion of Gemin5, and, more surprisingly, U1 does not require the U1 Sm-binding sequence. Instead, the ability of pre-U1 or mature U1 snRNAs to interact with SMN largely depends on the presence of U1-70K. Moreover, a mutant form of U1 lacking an Sm-binding site is entirely dependent on U1-70K for association with SMN. In contrast, mutant U1 snRNA defective in U1-70K binding, or with a strengthened Sm-binding-site sequence, requires Gemin5 for interaction with SMN. The authors5 have further shown that access to SMN, which is required for the maturation
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of multiple snRNAs, is competitive. They have found that knockdown of U1-70K, removing U1-specific SMN access, increases Sm-complex assembly on U2, U4 and U5 snRNAs at the expense of U1, presumably because SMN, which is normally occupied by U1, becomes available to the other snRNAs. The interaction between U1-70K and SMN is direct, requiring U1-70K amino acids 90–194 (which bind stem-loop 1 of U1 snRNA) and SMN’s C-terminal YG box (which is required for oligomerization). The authors have also touched upon the question of how the Sm core itself is built. Gemin2 has previously been shown to interact with and recruit Sm5 (ref. 6), an Sm assembly intermediate; however, formation of the complete Sm ring, Sm7, requires an Sm-binding site on RNA7. So et al.5 have shown that the N-terminal 99 amino acids of U1-70K, in combination with Gemin2, can form the complete Sm7 complex, a result consistent with observations made from recent SMN–Gemin2–Sm5 (ref. 8) and U1 snRNP crystal structures9,10. Thus, U1-70K helps mediate U1 assembly not only by helping it outcompete other snRNAs for access to SMN but also by recruiting the building blocks required by the SMN complex for snRNP maturation. Citing the unique pathways to SMN used by Gemin5 and U1-70K, So et al.5 have hypothesized 189
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© 2016 Nature America, Inc. All rights reserved.
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Figure 1 The SMN complex serves as a hub facilitating exchange of RNA cargos from a variety of RBP donors and acceptors and thus plays a central role in RNA metabolism. RNPs are symbolized by trucks carrying RNA cargos (shown as secondary structures) to and from the SMN complex. The Gemin2–SMN hub facilitates swapping of many RBPs between diverse RNA molecules. Gemin5 (red truck) is the canonical RBP donor for all snRNPs, bringing them to SMN so that they can acquire Sm rings. In contrast, U1-70K (pink) constitutes a U1-exclusive pathway to the SMN complex, thus allowing an alternative means for U1 to obtain an Sm ring.
that SMN serves as a hub (Fig. 1) that facilitates exchange of RNA cargo from a variety of RBP donors and acceptors and plays a central role in RNA metabolism. This hypothesis is consistent with observations that SMN is also able to form low-affinity nonspecific interactions with RNAs and that RBPs involved in diverse aspects of cellular metabolism—including heterogeneous nuclear RNP proteins, signalrecognition-particle components and transcription regulators—have been shown to bind SMN. The mapping by So et al.5 of the U1-70K– interacting region to the C-terminal YG-box domain of SMN is particularly interesting,
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given that the SMN isoform expressed in SMA patients lacks the majority of this domain11,12. In fact, the authors5 discuss how SMN’s role as a hub can account for the effects of RBP mutations in disease. SMN is the product of the SMA gene (SMN1), and the severity of the SMA disease correlates with both the extent of SMN deficiency and the corresponding decrease in assembly of the Sm core13,14. Furthermore, SMA is associated with a large number of widespread perturbations in RNA metabolism15, including alterations to alternative-splicing patterns and changes to transcript expression levels16. Although changes in splicing can be
easily attributed to a decreased abundance of core splicing factors, the other changes to RNA metabolism are better rationalized via SMN’s function in RNP exchange. Specifically, mutations in RBPs could weaken or strengthen interactions with SMN. Weakened binding would be detrimental because it would limit an RNP’s ability to acquire its proper cargo. Strengthened binding would not only delay a given RNP on the SMN complex but also restrict other RBPs in their ability to access SMN. Many open questions remain. Which RBPs interact with SMN? At what points in RNA processing do these RBP-SMN interactions occur? What regulates RNP exchange? Is SMN exchange affected by cellular stress or other physiological perturbations? How do diseases associated with RNA processing— such as SMA, frontotemporal lobar dementia or amyotrophic lateral sclerosis—affect SMN exchange? Answering these questions makes for exciting times ahead. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Valadkhan, S. & Gunawardane, L.S. Essays Biochem. 54, 79–90 (2013). 2. Brody, Y. et al. PLoS Biol. 9, e1000573 (2011). 3. Almada, A.E., Wu, X., Kriz, A.J., Burge, C.B. & Sharp, P.A. Nature 499, 360–363 (2013). 4. Berg, M.G. et al. Cell 150, 53–64 (2012). 5. So, B.R. et al. Nat. Struct. Mol. Biol. 23, 225–230 (2016). 6. Sarachan, K.L. et al. Biochem. J. 445, 361–370 (2012). 7. Raker, V.A., Hartmuth, K., Kastner, B. & Lührmann, R. Mol. Cell. Biol. 19, 6554–6565 (1999). 8. Zhang, R. et al. Cell 146, 384–395 (2011). 9. Kondo, Y., Oubridge, C., van Roon, A.-M.M. & Nagai, K. eLife 4, e04986 (2015). 10. Weber, G., Trowitzsch, S., Kastner, B., Lührmann, R. & Wahl, M.C. EMBO J. 29, 4172–4184 (2010). 11. Wan, L. et al. Mol. Cell. Biol. 25, 5543–5551 (2005). 12. Young, P.J. et al. Hum. Mol. Genet. 9, 2869–2877 (2000). 13. Burghes, A.H.M. & Beattie, C.E. Nat. Rev. Neurosci. 10, 597–609 (2009). 14. Lefebvre, S. et al. Nat. Genet. 16, 265–269 (1997). 15. Li, D.K., Tisdale, S., Lotti, F. & Pellizzoni, L. Semin. Cell Dev. Biol. 32, 22–29 (2014). 16. Zhang, Z. et al. Cell 133, 585–600 (2008).
volume 23 number 3 March 2016 nature structural & molecular biology
that this mechanism is not unique to the glutamate transporter family, and there may be many more transport proteins—unrelated in sequence and varied in biological function— that also use a twisting elevator to move their substrates across membranes. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Jardetzky, O. Nature 211, 969–970 (1966). 2. Yan, N. Annu. Rev. Biophys. 44, 257–283 (2015). 3. Reyes, N., Ginter, C. & Boudker, O. Nature 462, 880–885 (2009). 4. Mulligan, C. et al. Nat. Struct. Mol. Biol. 23, 256–263 (2016). 5. Coincon, M. et al. Nat. Struct. Mol. Biol. 23, 248–255 (2016).
6. Mancusso, R., Gregorio, G.G., Liu, Q. & Wang, D.N. Nature 491, 622–626 (2012). 7. Boudker, O., Ryan, R.M., Yernool, D., Shimamoto, K. & Gouaux, E. Nature 445, 387–393 (2007). 8. Crisman, T.J., Qu, S., Kanner, B.I. & Forrest, L.R. Proc. Natl. Acad. Sci. USA 106, 20752–20757 (2009). 9. Vergara-Jaque, A., Fenollar-Ferrer, C., Kaufmann, D. & Forrest, L.R. Front. Pharmacol. 6, 183 (2015). 10. Hunte, C. et al. Nature 435, 1197–1202 (2005). 11. Lee, C. et al. Nature 501, 573–577 (2013). 12. Lee, C. et al. J. Gen. Physiol. 144, 529–544 (2014). 13. Paulino, C., Wöhlert, D., Kapotova, E., Yildiz, Ö. & Kühlbrandt, W. eLife 3, e03583 (2014). 14. Bolla, J.R. et al. Nat. Commun. 6, 6874 (2015). 15. Su, C.C. et al. Cell Rep. 11, 61–70 (2015). 16. Hu, N.J., Iwata, S., Cameron, A.D. & Drew, D. Nature 478, 408–411 (2011). 17. Zhou, X. et al. Nature 505, 569–573 (2014). 18. Wohlert, D., Grotzinger, M.J., Kuhlbrandt, W. & Yildiz, O. eLife 4, (2015). 19. Slotboom, D.J. Nat. Rev. Microbiol. 12, 79–87 (2014).
npg
© 2016 Nature America, Inc. All rights reserved.
A new communication hub in the RNA world Megan Mayerle & Christine Guthrie During assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), the RNA-binding protein (RBP) Gemin5 recognizes the snRNP code and interacts with the large Gemin2–SMN complex. So et al. now find that Gemin2 also interacts with U1-70K, thereby conferring a preferential advantage on U1 snRNP assembly, and they extrapolate that SMN–Gemin2 serves a general ribonucleoprotein-exchange function.
The U1 snRNP has been best characterized regarding its role in precursor (pre)-mRNA splicing; however, its abundance in cells far exceeds that necessary for splicing regulation1. Furthermore, U1 is the only snRNP that localizes to intronless genes2, and it has been implicated in the regulation of transcription initiation as well as pre-mRNA length via its interactions with the polyadenylation machinery3,4. However, the mechanism through which cells maintain high levels of U1 relative to the other components of the spliceosome is not well understood. In this issue, So et al.5 reveal that small nuclear RNAs (snRNAs) compete for access to SMN, a protein complex required for snRNP maturation. They show that the unrelated RNA-binding protein (RBP) U1-70K gives U1 an advantage in this competition for SMN access, thus explaining the cellular overabundance of U1. Furthermore, these results hint that, rather than simply being a spliceosome-assembly factor, SMN may act as a hub for ribonucleoprotein (RNP) exchange, where a diverse set of RNPs swap their cargo Megan Mayerle and Christine Guthrie are at the Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. e-mail: [email protected]
RNAs (Fig. 1). These findings may also explain the widespread perturbations in RNA metabolism observed in the neurological disease spinal muscle atrophy (SMA). So et al.5 have established that U1 snRNA has two potential pathways to interact with SMN: (i) the canonical pathway used by all snRNAs, which requires the RBP Gemin5, and (ii) a unique pathway that makes use of U1-70K. Through a series of pulldown assays designed to assess the roles of U1 stem-loop 1 and the snRNP code in assembly of the Sm core, the authors have demonstrated that depletion of Gemin5 abolishes U4 snRNA’s ability to interact with the SMN complex, as expected. In contrast, the interaction between U1 snRNA and SMN is only modestly affected by depletion of Gemin5, and, more surprisingly, U1 does not require the U1 Sm-binding sequence. Instead, the ability of pre-U1 or mature U1 snRNAs to interact with SMN largely depends on the presence of U1-70K. Moreover, a mutant form of U1 lacking an Sm-binding site is entirely dependent on U1-70K for association with SMN. In contrast, mutant U1 snRNA defective in U1-70K binding, or with a strengthened Sm-binding-site sequence, requires Gemin5 for interaction with SMN. The authors5 have further shown that access to SMN, which is required for the maturation
nature structural & molecular biology volume 23 number 3 March 2016
of multiple snRNAs, is competitive. They have found that knockdown of U1-70K, removing U1-specific SMN access, increases Sm-complex assembly on U2, U4 and U5 snRNAs at the expense of U1, presumably because SMN, which is normally occupied by U1, becomes available to the other snRNAs. The interaction between U1-70K and SMN is direct, requiring U1-70K amino acids 90–194 (which bind stem-loop 1 of U1 snRNA) and SMN’s C-terminal YG box (which is required for oligomerization). The authors have also touched upon the question of how the Sm core itself is built. Gemin2 has previously been shown to interact with and recruit Sm5 (ref. 6), an Sm assembly intermediate; however, formation of the complete Sm ring, Sm7, requires an Sm-binding site on RNA7. So et al.5 have shown that the N-terminal 99 amino acids of U1-70K, in combination with Gemin2, can form the complete Sm7 complex, a result consistent with observations made from recent SMN–Gemin2–Sm5 (ref. 8) and U1 snRNP crystal structures9,10. Thus, U1-70K helps mediate U1 assembly not only by helping it outcompete other snRNAs for access to SMN but also by recruiting the building blocks required by the SMN complex for snRNP maturation. Citing the unique pathways to SMN used by Gemin5 and U1-70K, So et al.5 have hypothesized 189
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© 2016 Nature America, Inc. All rights reserved.
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Figure 1 The SMN complex serves as a hub facilitating exchange of RNA cargos from a variety of RBP donors and acceptors and thus plays a central role in RNA metabolism. RNPs are symbolized by trucks carrying RNA cargos (shown as secondary structures) to and from the SMN complex. The Gemin2–SMN hub facilitates swapping of many RBPs between diverse RNA molecules. Gemin5 (red truck) is the canonical RBP donor for all snRNPs, bringing them to SMN so that they can acquire Sm rings. In contrast, U1-70K (pink) constitutes a U1-exclusive pathway to the SMN complex, thus allowing an alternative means for U1 to obtain an Sm ring.
that SMN serves as a hub (Fig. 1) that facilitates exchange of RNA cargo from a variety of RBP donors and acceptors and plays a central role in RNA metabolism. This hypothesis is consistent with observations that SMN is also able to form low-affinity nonspecific interactions with RNAs and that RBPs involved in diverse aspects of cellular metabolism—including heterogeneous nuclear RNP proteins, signalrecognition-particle components and transcription regulators—have been shown to bind SMN. The mapping by So et al.5 of the U1-70K– interacting region to the C-terminal YG-box domain of SMN is particularly interesting,
190
given that the SMN isoform expressed in SMA patients lacks the majority of this domain11,12. In fact, the authors5 discuss how SMN’s role as a hub can account for the effects of RBP mutations in disease. SMN is the product of the SMA gene (SMN1), and the severity of the SMA disease correlates with both the extent of SMN deficiency and the corresponding decrease in assembly of the Sm core13,14. Furthermore, SMA is associated with a large number of widespread perturbations in RNA metabolism15, including alterations to alternative-splicing patterns and changes to transcript expression levels16. Although changes in splicing can be
easily attributed to a decreased abundance of core splicing factors, the other changes to RNA metabolism are better rationalized via SMN’s function in RNP exchange. Specifically, mutations in RBPs could weaken or strengthen interactions with SMN. Weakened binding would be detrimental because it would limit an RNP’s ability to acquire its proper cargo. Strengthened binding would not only delay a given RNP on the SMN complex but also restrict other RBPs in their ability to access SMN. Many open questions remain. Which RBPs interact with SMN? At what points in RNA processing do these RBP-SMN interactions occur? What regulates RNP exchange? Is SMN exchange affected by cellular stress or other physiological perturbations? How do diseases associated with RNA processing— such as SMA, frontotemporal lobar dementia or amyotrophic lateral sclerosis—affect SMN exchange? Answering these questions makes for exciting times ahead. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Valadkhan, S. & Gunawardane, L.S. Essays Biochem. 54, 79–90 (2013). 2. Brody, Y. et al. PLoS Biol. 9, e1000573 (2011). 3. Almada, A.E., Wu, X., Kriz, A.J., Burge, C.B. & Sharp, P.A. Nature 499, 360–363 (2013). 4. Berg, M.G. et al. Cell 150, 53–64 (2012). 5. So, B.R. et al. Nat. Struct. Mol. Biol. 23, 225–230 (2016). 6. Sarachan, K.L. et al. Biochem. J. 445, 361–370 (2012). 7. Raker, V.A., Hartmuth, K., Kastner, B. & Lührmann, R. Mol. Cell. Biol. 19, 6554–6565 (1999). 8. Zhang, R. et al. Cell 146, 384–395 (2011). 9. Kondo, Y., Oubridge, C., van Roon, A.-M.M. & Nagai, K. eLife 4, e04986 (2015). 10. Weber, G., Trowitzsch, S., Kastner, B., Lührmann, R. & Wahl, M.C. EMBO J. 29, 4172–4184 (2010). 11. Wan, L. et al. Mol. Cell. Biol. 25, 5543–5551 (2005). 12. Young, P.J. et al. Hum. Mol. Genet. 9, 2869–2877 (2000). 13. Burghes, A.H.M. & Beattie, C.E. Nat. Rev. Neurosci. 10, 597–609 (2009). 14. Lefebvre, S. et al. Nat. Genet. 16, 265–269 (1997). 15. Li, D.K., Tisdale, S., Lotti, F. & Pellizzoni, L. Semin. Cell Dev. Biol. 32, 22–29 (2014). 16. Zhang, Z. et al. Cell 133, 585–600 (2008).
volume 23 number 3 March 2016 nature structural & molecular biology
