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A structural dynamics model for how CPEB3 binding to SUMO2 can regulate translational control in dendritic spines [1]

['Xinyu Gu', 'Center For Theoretical Biological Physics', 'Rice University', 'Houston', 'Texas', 'United States Of America', 'Department Of Chemistry', 'Nicholas P. Schafer', 'Carlos Bueno', 'Wei Lu']

Date: 2022-11 

A prion-like RNA-binding protein, CPEB3, can regulate local translation in dendritic spines. CPEB3 monomers repress translation, whereas CPEB3 aggregates activate translation of its target mRNAs. However, the CPEB3 aggregates, as long-lasting prions, may raise the problem of unregulated translational activation. Here, we propose a computational model of the complex structure between CPEB3 RNA-binding domain (CPEB3-RBD) and small ubiquitin-like modifier protein 2 (SUMO2). Free energy calculations suggest that the allosteric effect of CPEB3-RBD/SUMO2 interaction can amplify the RNA-binding affinity of CPEB3. Combining with previous experimental observations on the SUMOylation mode of CPEB3, this model suggests an equilibrium shift of mRNA from binding to deSUMOylated CPEB3 aggregates to binding to SUMOylated CPEB3 monomers in basal synapses. This work shows how a burst of local translation in synapses can be silenced following a stimulation pulse, and explores the CPEB3/SUMO2 interplay underlying the structural change of synapses and the formation of long-term memories.

Local translation of specific synaptic proteins provides the molecular basis for the structural change in dendritic spines, which is essential for long-term memories. A functional prion-like RNA-binding protein, CPEB3, has been proposed as a synaptic tag to regulate local translation in dendritic spines. More interestingly, the soluble CPEB3 monomers repress translation, whereas the CPEB3 aggregates activate the translation of its target mRNAs. The CPEB3 aggregates, however, that act as long-lasting prions providing “conformational memory”, may raise the problem of translational activation being unregulated. Here, we propose a computational model of the complex structure between CPEB3 RNA-binding domain (CPEB3-RBD) and small ubiquitin-like modifier protein 2 (SUMO2). Free energy calculations suggest that the allosteric binding of CPEB3 with SUMO2 can confine the CPEB3-RBD to a conformation that favors RNA-binding, and thereby can amplify its RNA-binding affinity. Combining this model with previous experiments showing that CPEB3 monomers are SUMOylated in basal synapses but become deSUMOylated and start to aggregate upon stimulation, we suggest a way in which the translational control of CPEB3 can be switched back to a repressive mode after a stimulation pulse, through an RNA binding shift from binding to CPEB3 fibers to binding to SUMOylated CPEB3 monomers in basal synapses.

Funding: XG, NPS, CB, WL, and PGW were supported by the NSF Division of Chemistry RAISE grant CHE-1743392 and by the Center for Theoretical Biological Physics, sponsored by the NSF Division of Physics grant PHY-2019745. CB was also supported by the PoLS Student Research Network sponsored by the NSF Division of Physics grant 1522550. PGW was supported by the D. R. Bullard-Welch Chair at Rice University, Grant C-0016. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1 Introduction

The remodeling of the actin cytoskeleton in dendritic spines serves as the molecular basis for the structural changes of synapses [1] which are associated with the formation of long-term memories [2]. A synaptic tag is required to label budding synapses so as to regulate the local translation of actin mRNA and the mRNA of other synaptic proteins over long periods of time. A promising candidate for the synaptic tag is the mammalian cytoplasmic polyadenylation element-binding protein 3 (CPEB3). Mammalian CPEB3 [3] and its homologs, ApCPEB in Aplysia [4] and Orb2 in Drosophila [5, 6], have been shown to regulate the translation of synaptic proteins by binding with the U-rich CPE sequence in the 3’ untranslated region (UTR) of target mRNAs. Targets of CPEB3 include the message RNAs for actin [7] and other protein components essential for long-term synaptic persistence, like GluA1 and GluA2 [3]. The longevity of the synaptic tag is traced to the fact that, CPEB3 can form self-sustaining prion-like aggregates [8] that resist rapid molecular turnover [9]. Understanding the system dynamics of the CPEB3-actin regulation network [10] is essential to see how CPEB3 is able to consolidate synaptic structure and facilitate the formation of long-term memories.

CPEB3 in dendritic spines can be found either in a monomeric state or in an amyloid-like aggregated state. Intriguingly, the monomeric CPEB3 has been found to repress translation, while the aggregated form of CPEB3 activates the translation of its target mRNAs [11–13]. One idea that has been proposed is that monomeric CPEB3 localizes its target mRNAs through forming gel-like processing bodies (P bodies) in which translation is repressed and that CPEB3 aggregation into a prion form simply releases those mRNAs [13]. A structural study of a CPEB3 homolog, Orb2, has found, however, that Orb2 aggregated in the prion form still binds target mRNAs and interacts with various proteins that might further recruit translation promoting factors, like polyadenylation complex [14]. We have recently suggested that changes in the activation and repression of translation ability by CPEB3 can be explained by a vectorial channeling effect in which the recycling of ribosomes depends on the structure of CPEB3/RNA assemblies. [15]. Vectorial channeling arises from the vectorial nature of mRNA translation, along with the structurally polarized nature of the mRNA/prion assembly. This structural synergy allows CPEB3 aggregates to form a local translation factory assembly lines in which ribosomes are more efficiently recycled than they are by the monomeric form and thus turn on the translation of dormant target mRNAs. The CPEB3 aggregates, which function then as synaptic tags providing “conformational memory”, would be stable in synapses. Such stability, by itself would seemingly lead to continuous activation of local translation if aggregates were always found bound with their target mRNAs. This raises the question of whether and how such translational enhancement could be turned off so that synapses may return to a new basal state. (As illustrated in Fig 1).

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TIFF original image Download: Fig 1. The conformational changes of CPEB3 provide a mechanism for the switchable translational control in dendritic spines. In a basal state, monomeric CPEB3 (shown in red) is colocalized with P-bodies and represses the translation of its target mRNAs. In response to a stimulation signal, CPEB3 monomers are released from P-bodies and then aggregate into CPEB3 fibers (shown in green). CPEB3 fibers (using the vectorial channeling mechanism) activate the local translation of synaptic proteins, including the actin proteins which are the molecular basis for the growth of the spines. The stability of the remaining prion-like CPEB3 aggregates would raise a problem of how the translational activation is turned down after a stimulation pulse. SUMO binding provides a route to return to the new basal state. A legend is shown at the upper-right corner. https://doi.org/10.1371/journal.pcbi.1010657.g001

One possibility that has been suggested involves the SUMOylation of CPEB3. There is evidence that SUMOylation, a reversible post-translational modification, can regulate CPEB3 function. SUMO proteins, small ubiquitin-like modifier proteins, can be covalently attached to lysine residues of target proteins. After such SUMOylation, the SUMO modification can subsequently be deconjugated (deSUMOylation). Monomeric CPEB3 is found to be SUMOylated by SUMO2, one homolog of the SUMO protein family, and in that form it is soluble in basal synapses. After stimulation, CPEB3 becomes deSUMOylated and aggregates. [12] SUMOylation of CPEB3 has been found to facilitate its colocalization to P bodies and is crucial for repressing translation. [13] DeSUMOylation of CPEB3 exposes the prion-like domain (PRD) and the actin-binding domain (ABD) of CPEB3 and triggers actin-facilitated CPEB3 aggregation as a result. [16] An interesting fact in thinking about the system biology of SUMO2/CPEB3 is that SUMO2 mRNA itself is one of CPEB3’s targets. Therefore, there is a negative feedback loop between CPEB3 aggregation and SUMO2 synthesis [12]: The CPEB3 prion activates the translation of SUMO2 which can then be used for CPEB3 SUMOylation and lead to a return to translational repression. This negative feedback loop could thereby serve as a control on the balance between activation and repression of translation by CPEB3.

In this paper, we put forward a structural dynamics model of the interaction between SUMO2 and the RNA-binding domain (RBD) of SUMOylated CPEB3. We developed a structural model for the SUMO2/CPEB3-RBD complex through computational modeling using the Associative memory, Water-Mediated, Structure and Energy Model (AWSEM) [17, 18], a coarse-grained protein force field which has been optimized using energy landscape theory [19]. The AWSEM software has proved quite successful in predicting both monomeric protein structures and the structures of protein complexes. [17, 20] The SUMO2 protein simultaneously interacts with two distinct surfaces of the CPEB3 RNA-binding domain. By doing so, it closes the conformation of the RNA binding domain into a structure favorable for RNA binding. Using the AWSEM force field and combining it with the Three Sites Per Nucleotide model 2 (3SPN2), a coarse-grained force field for nucleic acids developed by the de Pablo group [21, 22], we have calculated the free energy profile for RNA dissociation from the SUMO2/CPEB3-RBD complex. These calculations show that the RNA-binding free energy for the SUMO2/CPEB3-RBD complex is 2 kcal/mol larger than that for isolated RBD in deSUMOylated CPEB3. We propose that the resulting difference in the RNA-binding affinity between the two forms causes a shift in the equilibrium of RNA binding from binding to the deSUMOylated CPEB3 aggregates to binding to SUMOylated CPEB3 monomers when synapses return to a new basal state. In this way, the translational control of CPEB3 becomes switchable in response to input signals: After stimulation, CPEB3 is deSUMOylated, so that CPEB3 aggregates and thereby activates translation of SUMO2 and other synaptic proteins. Monomeric CPEB3 once SUMOylated with newly synthesized SUMO2 proteins shifts the RNA-binding equilibrium, so that target mRNAs are recruited into P bodies by the SUMOylated CPEB3 and thereby are silenced.

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[1] Url: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1010657

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