- Email: [email protected]
Re-viewing the 3D Organization of mRNPs
Molecular Cell
Previews Re-viewing the 3D Organization of mRNPs Ge´rard Pierron1 and Dominique Weil2,* 1CNRS
UMR-9196, Institut Gustave Roussy, 94800 Villejuif, France Universite´, CNRS, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du De´veloppement, 75005 Paris, France *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2018.10.044 2Sorbonne
In this issue of Molecular Cell, using leading-edge technologies, Metkar et al. (2018) and Adivarahan et al. (2018) revisit the spatial organization of mRNPs, showing that they form flexible rod-like structures prior to translation that decompact during translation while the closed-loop conformation is rarely observed.
mRNA fate is governed by its combination of associated RNA-binding proteins (RBPs). In recent years, there has been major progress in the identification of RBPs, revealing that they correspond to a large fraction of the cell proteome (up to 10% in human; Hentze et al., 2018). However, elucidating all the molecular mechanisms underlying mRNA regulation represents a much more difficult task, in part due to the large variety of RBPs but also because of the myriad possible interactions between RBPs and between mRNAs and RBPs (Youn et al., 2018). That some RBPs are clearly sequence specific while others seem to be general regulators adds to the complexity. One key information that could enlighten the debate is the 3D organization of ribonucleoprotein complexes (mRNPs). In this issue of Molecular Cell, Metkar et al. (2018) and Adivarahan et al. (2018) make important contributions that renew our view of these complexes. How we picture mRNPs in space has considerably evolved with time (Figure 1). Starting from a naked unfolded mRNA molecule, it rapidly became an unfolded mRNA molecule bound to a few RBPs, then an mRNP where the mRNA locally adopts secondary structures and, in some cases, tertiary structures such as pseudoknots. Early knowledge on translation superimposed the presence of ribosomes to the picture, leading to the classical schematization of polysomes. Alongside, functional interactions between the 50 extremity of mRNAs and their 30 UTR were progressively found. Translation initiation is activated by the polyA-binding protein (PABP),
with eIF4G bridging PABP to the capbinding protein eIF4E, while ribosome recycling on the same mRNA requires some 50 -30 looping of the mRNA molecule. Furthermore, most translation repressors bind to the 30 UTR of mRNAs while regulating cap-dependent translation initiation. Similarly, regulators of the 50 mRNA decay machinery are recruited at the 30 extremity during deadenylation. The accumulation of such examples pushed our representation of mRNPs toward the most popular closed-loop model (first proposed by Alan Jacobson in 1996). However, while it has been a great source of inspiration for many years, imaging of such closed-loop mRNPs remained sparse. Electron microscopy (EM) analysis provided the first real images of mRNPs. Cytoplasmic polysomes adopt a striking organization in spirals and rosettes (Palade, 1955). In the nucleus, nascent mRNAs form fibrils of 5–7 nm diameter (Fakan and Puvion, 1980), while mature mRNPs can be highly compact, as exemplified by the gigantic Balbiani Ring mRNA (40 kb) of Chironomus tentans salivary glands, which forms a 40– 50 nm globular mRNP that transiently unfolds into a 20-nm-diameter rod-like particle to pass the nuclear pore (Stevens and Swift, 1966). Progressively, EM imaging entered a period of relative indifference. The observation in atomic force microscopy of circular RNAs formed in vitro in the presence of excess eIF4E, eIF4G1, and Pab1p (Wells et al., 1998) reinforced the closed-loop model, which became dominant. In this issue of Molecular Cell, the groups of Melissa Moore and Daniel Zen-
klusen tackled the issue of mRNP conformation using the latest developments of next-generation sequencing (NGS) (Metkar et al., 2018) and super-resolution microscopy imaging (Adivarahan et al., 2018). Metkar et al. (2018) developed an elegant method, called ‘‘RNA immunoprecipitation and proximity ligation in tandem’’ (RIPPLiT), to map the 3D organization of purified RNPs. Applying the method to the RNPs containing the exon junction complex (EJC), a complex that binds RNAs during splicing in the nucleus and that is released upon translation in the cytoplasm, they show for hundreds of transcripts that nascent and pre-translational mRNPs compact into a linearly organized flexible rod-like structure (Metkar et al., 2018). The caveat of this approach is that it does not provide any information on the size of the object, precluding any comparison with previous EM images of mRNPs. However, this genome-wide procedure, which in principle can apply to any RBP complex, reveals intramolecular proximities independently of their distance. One can anticipate that it will provide as much new information as Hi-C did for chromatin conformation. Adivarahan et al. (2018) combined single-molecule fluorescent in situ hybridization (smFISH) with two or three probe sets and structured illuminating microscopy (SIM) to measure the distance between 50 and 30 RNA ends in cells. The striking images obtained on three endogenous coding transcripts show that mRNPs are extended during translation and have distant 50 and 30 ends. In contrast, upon pharmacologically induced translation inhibition, mRNPs adopt a compact
Molecular Cell 72, November 15, 2018 ª 2018 Elsevier Inc. 603
Molecular Cell
Previews cap
1955
polyA
CBPs
PABPs e.g. IRE-BP
e.g. ARE-BPs
100 nm
ribosomes
1996 Closed-loop model untranslated
translaƟng
Pre-translaƟon 3’
5’
translaƟng
? 5’ 5’
2018
3’
Post-translaƟon
3’
Adivarahan et al. Mektar et al.
Figure 1. Spatial Organization of mRNPs Since mRNP discovery, their representations progressively gained in complexity, and a closed-loop model of repressed or actively translated mRNPs progressively emerged from functional studies. The upper right panel is an electron microscopy image of spiral polysomes in the cytoplasm of human cells. New results from Adivarahan et al. (2018) and Metkar et al. (2018) indicate that mRNPs are compact and flexible rod-like structures before translation, which decompact during translation and recompact to even greater extent following translation inhibition, questioning the closed-loop conformation as a representative state for translating mRNAs.
conformation with colocalized 50 and 30 ends (Adivarahan et al., 2018). The decompaction of translated mRNPs is consistent with both mRNA unfolding during translation elongation and extended polysomes seen in EM (Figure 1). In contrast, following translation inhibition, mRNPs are so compact that further studies will be required to understand whether they correspond to a folded rod-like structure and whether their 50 and 30 extremities interact. While the spatial resolution of the imaging is remarkable (21 nm, close to the ribosome diameter), one can dream of delineating the mRNP organization in yet more detail. However, while cryo-EM has become increasingly potent to solve the structure of homogeneous RNPs, such 604 Molecular Cell 72, November 15, 2018
as snRNPs and ribosomes, studying extremely heterogenous mRNPs is much more challenging. Remarkably and surprisingly, both studies come to the conclusion that the closed-loop model is not appropriate to describe mRNP conformation in cells, neither before (Metkar et al., 2018) nor during translation (Adivarahan et al., 2018). The observation of distant 50 and 30 ends is consistent with a previous study in yeast showing that most 50 and 30 mRNA ends do not co-purify after crosslinking (Archer et al., 2015). The same conclusion was reached by Roy Parker’s lab in a parallel study (Khong and Parker, 2018) using an approach similar to Adivarahan et al. (2018). After translation inhibition, they also observed that mRNPs
recompact, with 50 and 30 ends moving closer. Collectively, these data argue for a compact and flexible linear rod-like mRNP structure assembled in the nucleus, which unfolds upon translation elongation and maximally compacts after translation arrest. Thus, the closedloop conformation deduced from extensive functional and biochemical studies may only represent a transient step in translation initiation, while it would better describe EJC-free post-translated mRNPs. This re-viewing of mRNP conformation opens new possibilities in the field of post-transcriptional regulation. It will be key for the modeling of RNP granules that contain thousands of mRNP molecules, such as P-bodies and stress granules. These RNP condensates are now understood to form following liquid-liquid phase transition, a process that depends on physical parameters, such as temperature and salt, but also on the conformation of the condensed material. Based on EM, we reported that mRNPs seem fibrillar in P-bodies (Ernoult-Lange et al., 2012), which may represent yet another decompacted conformation not investigated here. More generally, the Moore and Zenklusen studies should inspire not only the revisiting of the mechanisms of 50 -30 functional interactions, but also the understanding of how the combinatorial association of RBPs, possibly through long-range interactions, turns to a coherent fine-tuning of mRNA translation and decay.
REFERENCES Adivarahan, S., Livingston, N., Nicholson, B., Rahman, S., Wu, B., Rissland, O.S., and Zenklusen, D. (2018). Spatial organization of single mRNPs at different stages of the gene expression pathway. Mol. Cell 72, this issue, 727–738. Archer, S.K., Shirokikh, N.E., Hallwirth, C.V., Beilharz, T.H., and Preiss, T. (2015). Probing the closed-loop model of mRNA translation in living cells. RNA Biol. 12, 248–254. Ernoult-Lange, M., Baconnais, S., Harper, M., Minshall, N., Souquere, S., Boudier, T., Be´nard, M., Andrey, P., Pierron, G., Kress, M., et al. (2012). Multiple binding of repressed mRNAs by the Pbody protein Rck/p54. RNA 18, 1702–1715. Fakan, S., and Puvion, E. (1980). The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int. Rev. Cytol. 65, 255–299.
Molecular Cell
Previews Hentze, M.W., Castello, A., Schwarzl, T., and Preiss, T. (2018). A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327–341. Khong, A., and Parker, R. (2018). mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction. J. Cell Biol. jcb.201806183. https://doi.org/10.1083/jcb. 201806183. Metkar, M., Ozadam, H., Lajoie, B.R., Imakaev, M., Mirny, L.A., Dekker, J., and Moore, M.J.
(2018). Higher-order organization principles of pre-translational mRNPs. Mol. Cell 72, this issue, 715–726. Palade, G.E. (1955). A small particulate component of the cytoplasm. J. Biophys. Biochem. Cytol. 1, 59–68. Stevens, B.J., and Swift, H. (1966). RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J. Cell Biol. 31, 55–77.
Wells, S.E., Hillner, P.E., Vale, R.D., and Sachs, A.B. (1998). Circularization of mRNA by eukaryotic translation initiation factors. Mol. Cell 2, 135–140. Youn, J.-Y., Dunham, W.H., Hong, S.J., Knight, J.D.R., Bashkurov, M., Chen, G.I., Bagci, H., Rathod, B., MacLeod, G., Eng, S.W.M., et al. (2018). High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies. Mol. Cell 69, 517–532.e11.
To Build by Destruction Lihui Wang1 and Yihong Ye1,* 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2018.10.043
In this issue of Molecular Cell, Weith et al. (2018) demonstrate that p97, together with a SEP adaptor, can catalyze ordered subunit exchange to facilitate the biogenesis of protein phosphatase-1 (PP1) holoenzyme, establishing a novel ubiquitin-independent ‘‘segregase’’ function for this versatile ATPase. Protein phosphatase 1 (PP1) is an essential serine/threonine phosphatase that catalyzes a substantial fraction of protein dephosphorylation events in eukaryotic cells. It functions as a collection of obligate holoenzyme complexes, each consisting of a distinct regulatory adaptor that confers substrate specificity and a catalytic subunit, also named PP1. Intriguingly, during the biogenesis of PP1 holoenzymes, newly synthesized PP1 is first held by two cofactors— SDS22 and inhibitor-3 (I3)—forming a stable trimeric complex with no activity. These inhibitory partners are later exchanged with one of the many substrate-specific adaptors to produce active PP1 holoenzymes (Peti et al., 2013). How subunit exchange is achieved during PP1 biogenesis has been unclear. In this issue of Molecular Cell, Weith et al. uncovered an energydependent protein-disassembling reaction as an important step in PP1 biogenesis. This process is catalyzed by the type II AAA (ATPase-associated with diverse cellular activities) ATPase p97, which disrupts the inhibitory PP1 complex to facilitate subunit exchange
and maturation of PP1 holoenzymes (Weith et al., 2018). p97 (also named VCP in mammals or CDC48 in yeast) is an abundant, highly conserved hexameric ATPase whose major function is to separate polypeptides from protein complexes, chromatin DNA, or organelle membranes in various cellular compartments (Ye et al., 2017). Like other type II AAA ATPases, p97 contains two similar AAA ATPase domains (termed D1 and D2), which oligomerize into two stacked rings with a central pore. It also has an N-terminal regulatory domain that interacts with a variety of cofactors. These cofactors promote substrate recruitment or processing and thus define a complex functional repertoire for p97/CDC48 (Ye et al., 2017). The best understood function of p97/ CDC48 is in endoplasmic reticulum-associated degradation (ERAD), in which it acts as an ER-associated, ubiquitindependent protein ‘‘unfoldase.’’ This function is assisted by the Ufd1-Npl4 dimeric cofactor complex, which uses several ubiquitin binding motifs for substrate recruitment. Once ubiquitinated polypeptides engage p97, it hydrolyzes
ATP, threading substrates through its central pore to cause their unfolding and separation from the ER membrane (Figure 1A) (Bodnar and Rapoport, 2017). Given the well-established ‘‘segregase’’ function, Weith et al. (2018) hypothesized that p97 may facilitate PP1 biogenesis by destabilizing the initial inhibitory complex. Using a pulse-chase approach, they first confirmed that newly synthesized PP1, SDS22, and I3 indeed form a transient complex whose disassembly allows PP1 to interact with a functional substrate specifier such as NIPP1. Strikingly, this highly coordinated subunit exchange process is prohibited when p97 is inactivated by either siRNA-mediated gene silencing or p97-specific inhibitors. To elucidate the molecular mechanism of this p97-dependent process, Weith et al. (2018) focused on a class of p97 adaptors named SEP (for Shp1, eyesclosed, and p47) domain-containing adaptors because genetic and biochemical studies in yeast have implicated the SEP-containing protein Shp1 in PP1 biogenesis (Zhang et al., 1995). They first showed, by co-immunoprecipitation, that among the four mammalian SEP
Molecular Cell 72, November 15, 2018 ª 2018 Elsevier Inc. 605
Previews Re-viewing the 3D Organization of mRNPs Ge´rard Pierron1 and Dominique Weil2,* 1CNRS
UMR-9196, Institut Gustave Roussy, 94800 Villejuif, France Universite´, CNRS, Institut de Biologie Paris-Seine (IBPS), Laboratoire de Biologie du De´veloppement, 75005 Paris, France *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2018.10.044 2Sorbonne
In this issue of Molecular Cell, using leading-edge technologies, Metkar et al. (2018) and Adivarahan et al. (2018) revisit the spatial organization of mRNPs, showing that they form flexible rod-like structures prior to translation that decompact during translation while the closed-loop conformation is rarely observed.
mRNA fate is governed by its combination of associated RNA-binding proteins (RBPs). In recent years, there has been major progress in the identification of RBPs, revealing that they correspond to a large fraction of the cell proteome (up to 10% in human; Hentze et al., 2018). However, elucidating all the molecular mechanisms underlying mRNA regulation represents a much more difficult task, in part due to the large variety of RBPs but also because of the myriad possible interactions between RBPs and between mRNAs and RBPs (Youn et al., 2018). That some RBPs are clearly sequence specific while others seem to be general regulators adds to the complexity. One key information that could enlighten the debate is the 3D organization of ribonucleoprotein complexes (mRNPs). In this issue of Molecular Cell, Metkar et al. (2018) and Adivarahan et al. (2018) make important contributions that renew our view of these complexes. How we picture mRNPs in space has considerably evolved with time (Figure 1). Starting from a naked unfolded mRNA molecule, it rapidly became an unfolded mRNA molecule bound to a few RBPs, then an mRNP where the mRNA locally adopts secondary structures and, in some cases, tertiary structures such as pseudoknots. Early knowledge on translation superimposed the presence of ribosomes to the picture, leading to the classical schematization of polysomes. Alongside, functional interactions between the 50 extremity of mRNAs and their 30 UTR were progressively found. Translation initiation is activated by the polyA-binding protein (PABP),
with eIF4G bridging PABP to the capbinding protein eIF4E, while ribosome recycling on the same mRNA requires some 50 -30 looping of the mRNA molecule. Furthermore, most translation repressors bind to the 30 UTR of mRNAs while regulating cap-dependent translation initiation. Similarly, regulators of the 50 mRNA decay machinery are recruited at the 30 extremity during deadenylation. The accumulation of such examples pushed our representation of mRNPs toward the most popular closed-loop model (first proposed by Alan Jacobson in 1996). However, while it has been a great source of inspiration for many years, imaging of such closed-loop mRNPs remained sparse. Electron microscopy (EM) analysis provided the first real images of mRNPs. Cytoplasmic polysomes adopt a striking organization in spirals and rosettes (Palade, 1955). In the nucleus, nascent mRNAs form fibrils of 5–7 nm diameter (Fakan and Puvion, 1980), while mature mRNPs can be highly compact, as exemplified by the gigantic Balbiani Ring mRNA (40 kb) of Chironomus tentans salivary glands, which forms a 40– 50 nm globular mRNP that transiently unfolds into a 20-nm-diameter rod-like particle to pass the nuclear pore (Stevens and Swift, 1966). Progressively, EM imaging entered a period of relative indifference. The observation in atomic force microscopy of circular RNAs formed in vitro in the presence of excess eIF4E, eIF4G1, and Pab1p (Wells et al., 1998) reinforced the closed-loop model, which became dominant. In this issue of Molecular Cell, the groups of Melissa Moore and Daniel Zen-
klusen tackled the issue of mRNP conformation using the latest developments of next-generation sequencing (NGS) (Metkar et al., 2018) and super-resolution microscopy imaging (Adivarahan et al., 2018). Metkar et al. (2018) developed an elegant method, called ‘‘RNA immunoprecipitation and proximity ligation in tandem’’ (RIPPLiT), to map the 3D organization of purified RNPs. Applying the method to the RNPs containing the exon junction complex (EJC), a complex that binds RNAs during splicing in the nucleus and that is released upon translation in the cytoplasm, they show for hundreds of transcripts that nascent and pre-translational mRNPs compact into a linearly organized flexible rod-like structure (Metkar et al., 2018). The caveat of this approach is that it does not provide any information on the size of the object, precluding any comparison with previous EM images of mRNPs. However, this genome-wide procedure, which in principle can apply to any RBP complex, reveals intramolecular proximities independently of their distance. One can anticipate that it will provide as much new information as Hi-C did for chromatin conformation. Adivarahan et al. (2018) combined single-molecule fluorescent in situ hybridization (smFISH) with two or three probe sets and structured illuminating microscopy (SIM) to measure the distance between 50 and 30 RNA ends in cells. The striking images obtained on three endogenous coding transcripts show that mRNPs are extended during translation and have distant 50 and 30 ends. In contrast, upon pharmacologically induced translation inhibition, mRNPs adopt a compact
Molecular Cell 72, November 15, 2018 ª 2018 Elsevier Inc. 603
Molecular Cell
Previews cap
1955
polyA
CBPs
PABPs e.g. IRE-BP
e.g. ARE-BPs
100 nm
ribosomes
1996 Closed-loop model untranslated
translaƟng
Pre-translaƟon 3’
5’
translaƟng
? 5’ 5’
2018
3’
Post-translaƟon
3’
Adivarahan et al. Mektar et al.
Figure 1. Spatial Organization of mRNPs Since mRNP discovery, their representations progressively gained in complexity, and a closed-loop model of repressed or actively translated mRNPs progressively emerged from functional studies. The upper right panel is an electron microscopy image of spiral polysomes in the cytoplasm of human cells. New results from Adivarahan et al. (2018) and Metkar et al. (2018) indicate that mRNPs are compact and flexible rod-like structures before translation, which decompact during translation and recompact to even greater extent following translation inhibition, questioning the closed-loop conformation as a representative state for translating mRNAs.
conformation with colocalized 50 and 30 ends (Adivarahan et al., 2018). The decompaction of translated mRNPs is consistent with both mRNA unfolding during translation elongation and extended polysomes seen in EM (Figure 1). In contrast, following translation inhibition, mRNPs are so compact that further studies will be required to understand whether they correspond to a folded rod-like structure and whether their 50 and 30 extremities interact. While the spatial resolution of the imaging is remarkable (21 nm, close to the ribosome diameter), one can dream of delineating the mRNP organization in yet more detail. However, while cryo-EM has become increasingly potent to solve the structure of homogeneous RNPs, such 604 Molecular Cell 72, November 15, 2018
as snRNPs and ribosomes, studying extremely heterogenous mRNPs is much more challenging. Remarkably and surprisingly, both studies come to the conclusion that the closed-loop model is not appropriate to describe mRNP conformation in cells, neither before (Metkar et al., 2018) nor during translation (Adivarahan et al., 2018). The observation of distant 50 and 30 ends is consistent with a previous study in yeast showing that most 50 and 30 mRNA ends do not co-purify after crosslinking (Archer et al., 2015). The same conclusion was reached by Roy Parker’s lab in a parallel study (Khong and Parker, 2018) using an approach similar to Adivarahan et al. (2018). After translation inhibition, they also observed that mRNPs
recompact, with 50 and 30 ends moving closer. Collectively, these data argue for a compact and flexible linear rod-like mRNP structure assembled in the nucleus, which unfolds upon translation elongation and maximally compacts after translation arrest. Thus, the closedloop conformation deduced from extensive functional and biochemical studies may only represent a transient step in translation initiation, while it would better describe EJC-free post-translated mRNPs. This re-viewing of mRNP conformation opens new possibilities in the field of post-transcriptional regulation. It will be key for the modeling of RNP granules that contain thousands of mRNP molecules, such as P-bodies and stress granules. These RNP condensates are now understood to form following liquid-liquid phase transition, a process that depends on physical parameters, such as temperature and salt, but also on the conformation of the condensed material. Based on EM, we reported that mRNPs seem fibrillar in P-bodies (Ernoult-Lange et al., 2012), which may represent yet another decompacted conformation not investigated here. More generally, the Moore and Zenklusen studies should inspire not only the revisiting of the mechanisms of 50 -30 functional interactions, but also the understanding of how the combinatorial association of RBPs, possibly through long-range interactions, turns to a coherent fine-tuning of mRNA translation and decay.
REFERENCES Adivarahan, S., Livingston, N., Nicholson, B., Rahman, S., Wu, B., Rissland, O.S., and Zenklusen, D. (2018). Spatial organization of single mRNPs at different stages of the gene expression pathway. Mol. Cell 72, this issue, 727–738. Archer, S.K., Shirokikh, N.E., Hallwirth, C.V., Beilharz, T.H., and Preiss, T. (2015). Probing the closed-loop model of mRNA translation in living cells. RNA Biol. 12, 248–254. Ernoult-Lange, M., Baconnais, S., Harper, M., Minshall, N., Souquere, S., Boudier, T., Be´nard, M., Andrey, P., Pierron, G., Kress, M., et al. (2012). Multiple binding of repressed mRNAs by the Pbody protein Rck/p54. RNA 18, 1702–1715. Fakan, S., and Puvion, E. (1980). The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int. Rev. Cytol. 65, 255–299.
Molecular Cell
Previews Hentze, M.W., Castello, A., Schwarzl, T., and Preiss, T. (2018). A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327–341. Khong, A., and Parker, R. (2018). mRNP architecture in translating and stress conditions reveals an ordered pathway of mRNP compaction. J. Cell Biol. jcb.201806183. https://doi.org/10.1083/jcb. 201806183. Metkar, M., Ozadam, H., Lajoie, B.R., Imakaev, M., Mirny, L.A., Dekker, J., and Moore, M.J.
(2018). Higher-order organization principles of pre-translational mRNPs. Mol. Cell 72, this issue, 715–726. Palade, G.E. (1955). A small particulate component of the cytoplasm. J. Biophys. Biochem. Cytol. 1, 59–68. Stevens, B.J., and Swift, H. (1966). RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J. Cell Biol. 31, 55–77.
Wells, S.E., Hillner, P.E., Vale, R.D., and Sachs, A.B. (1998). Circularization of mRNA by eukaryotic translation initiation factors. Mol. Cell 2, 135–140. Youn, J.-Y., Dunham, W.H., Hong, S.J., Knight, J.D.R., Bashkurov, M., Chen, G.I., Bagci, H., Rathod, B., MacLeod, G., Eng, S.W.M., et al. (2018). High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies. Mol. Cell 69, 517–532.e11.
To Build by Destruction Lihui Wang1 and Yihong Ye1,* 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2018.10.043
In this issue of Molecular Cell, Weith et al. (2018) demonstrate that p97, together with a SEP adaptor, can catalyze ordered subunit exchange to facilitate the biogenesis of protein phosphatase-1 (PP1) holoenzyme, establishing a novel ubiquitin-independent ‘‘segregase’’ function for this versatile ATPase. Protein phosphatase 1 (PP1) is an essential serine/threonine phosphatase that catalyzes a substantial fraction of protein dephosphorylation events in eukaryotic cells. It functions as a collection of obligate holoenzyme complexes, each consisting of a distinct regulatory adaptor that confers substrate specificity and a catalytic subunit, also named PP1. Intriguingly, during the biogenesis of PP1 holoenzymes, newly synthesized PP1 is first held by two cofactors— SDS22 and inhibitor-3 (I3)—forming a stable trimeric complex with no activity. These inhibitory partners are later exchanged with one of the many substrate-specific adaptors to produce active PP1 holoenzymes (Peti et al., 2013). How subunit exchange is achieved during PP1 biogenesis has been unclear. In this issue of Molecular Cell, Weith et al. uncovered an energydependent protein-disassembling reaction as an important step in PP1 biogenesis. This process is catalyzed by the type II AAA (ATPase-associated with diverse cellular activities) ATPase p97, which disrupts the inhibitory PP1 complex to facilitate subunit exchange
and maturation of PP1 holoenzymes (Weith et al., 2018). p97 (also named VCP in mammals or CDC48 in yeast) is an abundant, highly conserved hexameric ATPase whose major function is to separate polypeptides from protein complexes, chromatin DNA, or organelle membranes in various cellular compartments (Ye et al., 2017). Like other type II AAA ATPases, p97 contains two similar AAA ATPase domains (termed D1 and D2), which oligomerize into two stacked rings with a central pore. It also has an N-terminal regulatory domain that interacts with a variety of cofactors. These cofactors promote substrate recruitment or processing and thus define a complex functional repertoire for p97/CDC48 (Ye et al., 2017). The best understood function of p97/ CDC48 is in endoplasmic reticulum-associated degradation (ERAD), in which it acts as an ER-associated, ubiquitindependent protein ‘‘unfoldase.’’ This function is assisted by the Ufd1-Npl4 dimeric cofactor complex, which uses several ubiquitin binding motifs for substrate recruitment. Once ubiquitinated polypeptides engage p97, it hydrolyzes
ATP, threading substrates through its central pore to cause their unfolding and separation from the ER membrane (Figure 1A) (Bodnar and Rapoport, 2017). Given the well-established ‘‘segregase’’ function, Weith et al. (2018) hypothesized that p97 may facilitate PP1 biogenesis by destabilizing the initial inhibitory complex. Using a pulse-chase approach, they first confirmed that newly synthesized PP1, SDS22, and I3 indeed form a transient complex whose disassembly allows PP1 to interact with a functional substrate specifier such as NIPP1. Strikingly, this highly coordinated subunit exchange process is prohibited when p97 is inactivated by either siRNA-mediated gene silencing or p97-specific inhibitors. To elucidate the molecular mechanism of this p97-dependent process, Weith et al. (2018) focused on a class of p97 adaptors named SEP (for Shp1, eyesclosed, and p47) domain-containing adaptors because genetic and biochemical studies in yeast have implicated the SEP-containing protein Shp1 in PP1 biogenesis (Zhang et al., 1995). They first showed, by co-immunoprecipitation, that among the four mammalian SEP
Molecular Cell 72, November 15, 2018 ª 2018 Elsevier Inc. 605