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Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting an...
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... mutants had a normal short-term memory (Table S5D) and a strong detriment in long-term memory in comparison to the wild-type flies (3, orb2 DA , LI = 12.69; 1, orb2 + , LI = 30.31), almost as severe as mutants lacking the Q domain in both isoforms (2, orb2 DQ , LI = 2.15), suggesting that Orb2A function is critically required for long-term memory (Fig- ure 3; Table S4). However, these mutant flies were able to form residual but statistically significant memory likely to be mediated by Orb2B. ...Context 2
... assess the role of the Q domain in Orb2 isoforms, we gener- ated a specific deletion of this domain by reinserting into orb2 attP a genomic fragment in which disruption of either Orb2A or Orb2B was combined with the deletion of the Q domain. orb2 DQDA males, expressing only Orb2B lacking its Q domain, had a normal short-term memory (Table S5D) but, like orb2 DQ mutants, almost no long-term memory (4, orb2 DQDA , LI = 5.16; 2, orb2 DQ , LI = 2.15) ( Figure 3; Table S4), suggesting that the residual memory of the orb2 DA mutants might be mediated by the Q domain of Orb2B. ...Context 3
... the orb2 DQDB mutation was lethal when homozygous, we tested this allele in combination with the viable orb2 DA allele. These flies, which lack the Q domain specifically in Orb2A, had a normal short-term memory (Table S5D) but no long-term memory (5, orb2 DQDB /orb2 DA , LI = 2.86) ( Figure 3; Table S4). This lack of memory shows that the Q domain in Orb2A is essen- tial, and that of Orb2B insufficient, for long-term memory. ...Context 4
... learning index of these mutants was indistinguishable from control flies in which both isoforms are intact (6, orb2 DB /orb2 DQDA , LI = 16.97; 7, orb2 DB /orb2 DA LI = 20.83) ( Figure 3; Table S4). These results indicate that Orb2A has a specific role in long-term memory that requires the Q domain, which in Orb2B is both dispensable and insufficient. ...Context 5
... in contrast, appears to be more broadly and highly expressed and may mediate a more general function of Orb2 in development ( Cziko et al., 2009;Hafer et al., 2011;Richter, 2007;Shieh and Bonini, 2011). Orb2 has been reported to be present in the messenger RNPs, as we have also observed here specifically for Orb2B ( Figure S3), and is thought to control mRNA transport and translational repression ( Cziko et al., 2009;Mendez and Richter, 2001). During learning, Orb2A might interact with Orb2B-containing RNPs at the relevant synapses, releasing the associated mRNAs from translational repression or possibly even converting Orb2B from a translation repressor to an activator. ...Citations
... From a candidate genetic screen, we identify the conserved CPEB protein Orb2 as a specific regulator of rare-codon-biased protein expression in neurons and in spermatids of the testis. These findings mirror Orb2's known roles in both neuronal function [53][54][55][56][57][58][59][60] and in male fertility [61][62][63] . Using RNA sequencing, we then reveal that Orb2-upregulated brain mRNAs with abundant Orb2 binding sites have a rare-codon bias. ...
Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.
... One key mechanism involved in the regulation of the persistence of memory is the prion-like activity of CPEB in Aplysia [16,17,[25][26][27]81] and Orb2 in Drosophila [18,40,41,43,82]. In this work, we start with an immunoprecipitation screen and identify interactors belonging to the Hsp40 and Hsp70 families of proteins. ...
Orb2 the Drosophila homolog of cytoplasmic polyadenylation element binding (CPEB) protein forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation is dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A’s prion-like oligomerization forms long-term memory but fails to maintain it over time. Since this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here, we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Among these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, Mrj knockout exhibits a deficit in long-term memory and our observations suggest Mrj is needed in mushroom body neurons for the regulation of long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating ribosomes.
... 55 Furthermore, g-KCs output are required also in non-social associative visual learning. 57 Their similar roles in olfactory and visual learning, 14,16,18,24,52,58 as well as in mate copying (this study), and reacting to courtship conditioning 59,60 show that both, visual and olfactory cues of social or non-social origin elicit the functionality of MB g-neurons. Thus, these neurons appear to constitute a hub in the neuronal pathways of a large series of types of Drosophila associative learning. ...
Mate choice constitutes a major fitness-affecting decision often involving social learning leading to copying the preference of other individuals (i.e., mate copying). While mate copying exists in many taxa, its underlying neurobiological mechanisms remain virtually unknown. Here, we show in Drosophila melanogaster that the rutabaga gene is necessary to support mate copying. Rutabaga encodes an adenylyl cyclase (AC-Rut+) acting as a coincidence detector in associative learning. Since the brain localization requirements for AC-Rut+ expression differ in classical and operant learning, we determine the functional localization of AC-Rut+ for mate copying by artificially rescuing the expression of AC-Rut+ in neural subsets of a rutabaga mutant. We found that AC-Rut+ has to be expressed in the mushroom bodies’ Kenyon cells (KCs), specifically in the γ-KCs subset. Thus, this form of discriminative social learning requires the same KCs as non-social Pavlovian learning, suggesting that pathways of social and asocial learning overlap significantly.
... There is less conservation or agreement regarding sequence recognition by members of the CPEB2 subfamily in comparison to that of CPEB1, but these also seem to have affinity for U-rich elements [63][64][65]. Evidence exists for co-regulation of some targets of cytoplasmic polyadenylation by members of both CPEB subfamilies [60,[66][67][68], as well as for functions for CPEBs that are independent of modulation of poly(A)-tail length [69][70][71][72]. However, the majority of studies show that the central role of CPEBs is in regulating specific subsets of mRNA via . ...
Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3prime-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element-Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in non-animal lineages. Whether cytoplasmic polyadenylation is present in non-bilaterian animals (i.e., sponges, ctenophores, placozoans, cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.
... The RNA-binding protein Orb2 of Drosophila melanogaster also functions in neurons in an amyloid form (Hervás et al. 2020). Orb2 belongs to the CPEB (cytoplasmic polyadenylation element-binding) family of conserved regulatory mRNA-binding proteins that play an important role in the formation and maintenance of long-term memory in various animal species, from Aplysia californica gastropod to mammals (Si et al. 2010;Krüttner et al. 2012;Fioriti et al. 2015;Hervás et al. 2021). Some fragments of the California sea hare, mouse, and human CPEB family proteins are capable of forming amyloid fibrils in vitro (Stephan et al. 2015;Hervás et al. 2021;Flores et al. 2022). ...
Functional amyloids have been identified in a wide variety of organisms including bacteria, fungi, plants, and vertebrates. Intracellular and extracellular amyloid fibrils of different proteins perform storage, protective, structural, and regulatory functions. The structural organization of amyloid fibrils determines their unique physical and biochemical properties. The formation of these fibrillar structures can provide adaptive advantages that are picked up by natural selection. Despite the great interest in functional and pathological amyloids, questions about the conservatism of the amyloid properties of proteins and the regularities in the appearance of these fibrillar structures in evolution remain almost unexplored. Using bioinformatics approaches and summarizing the data published previously, we have shown that amyloid fibrils performing similar functions in different organisms have been arising repeatedly and independently in the course of evolution. On the other hand, we show that the amyloid properties of a number of bacterial and eukaryotic proteins are evolutionarily conserved. We also discuss the role of protein-based inheritance in the evolution of microorganisms. Considering that missense mutations and the emergence of prions cause the same consequences, we propose the concept that the formation of prions, similarly to mutations, generally causes a negative effect, although it can also lead to adaptations in rare cases. In general, our analysis revealed certain patterns in the emergence and spread of amyloid fibrillar structures in the course of evolution.
... Both isoforms have polyglutamine (polyQ), RNA-binding, and zinc finger domains, but differ in their N termini [7]. Orb2B is widely distributed in the Drosophila brain, while Orb2A is thought to be largely restricted to the synaptic zone of the mushroom body neurons [8,19]. Both of the Orb2 isoforms are essential for the formation of LTM; however, they are believed to have different functions, because the polyQ domain of Orb2A and the RNA-binding domain of Orb2B are essential for LTM. ...
... We found no significant difference in courtship index between orb2 R and WT males in the case of short-and mid-term memory ( Figure 1D). The results are consistent with published data that a complete deletion of the orb2 gene, a deletion of Orb2A (orb2 ΔA ), or a deletion of the polyglutamine domain of the Orb2 protein (orb2 ΔQ ) have no effect on the formation of short-or mid-term memory [19]. ...
... As shown in Figure 1E, orb2 R males had a pronounced deficit in LTM; their courtship index was comparable to that of naïve untrained WT males. Thus, the effects of the 3′UTR deletion are similar to those observed for the orb2 ΔA and orb2 ΔQ mutations [7,19]: no alteration occurred in short-term or mid-term memory, but LTM was strongly impaired. ...
Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3’UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3′UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3′UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.
... 150 Memory formation and storage by Orb2 is only possible by having the distinct roles of each isoform accessible through and conveyed by the unique heteromeric amyloid fold. 20,141,[144][145] These amyloid-dependent mechanisms for memory consolidation are conserved and similarly crucial within the Orb2 orthologues, Aplysia CPEB (ApCPEB) and the mammalian protein, CPEB3. 151 The assembly of Orb2 from its a-helix rich structure to the b-sheet aggregate has been found to be required for memory consolidation, [144][145] and is dependent on a His/Gln-rich prion-like domain (PLD) of 31 residues, as resolved by cryo-EM of Orb2 extracted from Drosophila heads. ...
... 20,141,[144][145] These amyloid-dependent mechanisms for memory consolidation are conserved and similarly crucial within the Orb2 orthologues, Aplysia CPEB (ApCPEB) and the mammalian protein, CPEB3. 151 The assembly of Orb2 from its a-helix rich structure to the b-sheet aggregate has been found to be required for memory consolidation, [144][145] and is dependent on a His/Gln-rich prion-like domain (PLD) of 31 residues, as resolved by cryo-EM of Orb2 extracted from Drosophila heads. 20 Similarly, Gln-rich domains are also present and implicated in memory formation for ApCPEB and CPEB3. ...
... 142 In particular, the Gln-rich domain of Orb2A, but not Orb2B, was found to be absolutely required for long-term memory storage, whilst the RNA-binding domain was the crucial aspect found for Orb2B. 144 Histidine residues in particular are believed to be important in the ability for this protein to associate and dissociate. At pH 4, the structure is largely a-helical and disordered, however once histidine residues are neutralised, the repellent charges are reduced and amyloid formation can occur. ...
Functional amyloids are a rapidly expanding class of fibrillar protein structures, with a core cross-β scaffold, where novel and advantageous biological function is generated by the assembly of the amyloid. The growing number of amyloid structures determined at high resolution reveal how this supramolecular template both accommodates a wide variety of amino acid sequences and also imposes selectivity on the assembly process. The amyloid fibril can no longer be considered a generic aggregate, even when associated with disease and loss of function. In functional amyloids the polymeric β-sheet rich structure provides multiple different examples of unique control mechanisms and structures that are finely tuned to deliver assembly or disassembly in response to physiological or environmental cues. Here we review the range of mechanisms at play in natural, functional amyloids, where tight control of amyloidogenicity is achieved by environmental triggers of conformational change, proteolytic generation of amyloidogenic fragments, or heteromeric seeding and amyloid fibril stability. In the amyloid fibril form, activity can be regulated by pH, ligand binding and higher order protofilament or fibril architectures that impact the arrangement of associated domains and amyloid stability. The growing understanding of the molecular basis for the control of structure and functionality delivered by natural amyloids in nearly all life forms should inform the development of therapies for amyloid-associated diseases and guide the design of innovative biomaterials.
... This residue is specific to only Orb2A and is not present in Orb2B. Thirdly, genetic deletion experiments suggested Orb2A does not need its RNA binding domain and Orb2B does not need its prion-like domain for the maintenance of memory (Krüttner et al., 2012). Finally, the addition of the prion-like domain of Orb2A can seed monomeric Orb2B to oligomerize and result in its change to a translational activator (Khan et al., 2015). ...
... One key mechanism involved in the regulation of the persistence of memory is the prion-like activity of CPEB in Aplysia (Heinrich and Lindquist, 2011;Miniaci et al., 2008;Raveendra et al., 2013;Si et al., 2010Si et al., , 2003aSi et al., , 2003b and Orb2 in Drosophila (Keleman et al., 2007;Khan et al., 2015;Krüttner et al., 2015Krüttner et al., , 2012Majumdar et al., 2012). In this work, we identify Mrj as a protein that is regulating the conversion of Orb2A from non-prion to prion-like oligomeric form. ...
Orb2 the Drosophila homolog of Cytoplasmic polyadenylation element binding protein (CPEB) forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation are dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A's prion-like oligomerization forms long-term memory but fails to maintain it over time. Since, this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Amongst these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, the knockdown of Mrj in the mushroom body neurons results in a deficit in long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating polysomes.
... The Drosophila homolog Orb2 has been well characterized, revealing an intriguing aggregation mechanism (25)(26)(27)(28)(29)(30)(31)(32). Two protein isoforms are expressed from the orb2 gene: ...
... Orb2A, which is highly aggregation-prone and kept at extremely low concentration in the resting-state synapse, and Orb2B, which is more soluble and makes up the majority of expressed Orb2 protein. Following synaptic stimulation, Orb2A forms stable aggregates that nucleate Orb2B amyloid formation, thereby switching Orb2 from a translation inhibitor to activator (27)(28)(29), possibly through recruitment of Orb2 monomer-or amyloid-specific binding partners that facilitate RNA degradation or translation, respectively (29,30). Both Orb2 isoform sequences are nearly identical, consisting of a Q/H-rich region and C-terminal RRMs. ...
... Both Orb2 isoform sequences are nearly identical, consisting of a Q/H-rich region and C-terminal RRMs. The isoforms differ only at the N-terminus: whereas Orb2B has a 162-residue serine/glycine-rich N-terminus that is predicted to be intrinsically disordered and as of yet has unknown function, the Orb2A Nterminus is only 9-residues in length, but is nevertheless critical for its self-assembly (27) and function in initiating Orb2B aggregation (28). ...
Amyloid protein aggregation is commonly associated with progressive neurodegenerative diseases, however not all amyloid fibrils are pathogenic. The neuronal cytoplasmic polyadenylation element binding (CPEB) protein is a regulator of synaptic mRNA translation, and has been shown to form functional amyloid aggregates that stabilize long-term memory. In adult Drosophila neurons, the CPEB homolog Orb2 is expressed as two isoforms, of which the Orb2B isoform is far more abundant, but the rarer Orb2A isoform is required to initiate Orb2 aggregation. The N-terminus is a distinctive feature of the Orb2A isoform and is critical for its aggregation. Intriguingly, replacement of phenylalanine in the 5th position of Orb2A with tyrosine (F5Y) in Drosophila impairs stabilization of long-term memory. The structure of endogenous Orb2B fibers was recently determined by cryo-EM, but the structure adopted by fibrillar Orb2A is less certain. Here we use micro-electron diffraction to determine the structure of the first nine N-terminal residues of Orb2A, at a resolution of 1.05 Å. We find that this segment (which we term M9I) forms an amyloid-like array of parallel in-register β-sheets, which interact through side chain interdigitation of aromatic and hydrophobic residues. Our structure provides an explanation for the decreased aggregation observed for the F5Y mutant, and offers a hypothesis for how the addition of a single atom (the tyrosyl oxygen) affects long-term memory. We also propose a structural model of Orb2A that integrates our structure of the M9I segment with the published Orb2B cryo-EM structure.
... Moreover, at physiological concentrations, amyloids are the most stable conformational state for many proteins, meaning that the native state is often a metastable phenomenon (Baldwin et al., 2011;Varela et al., 2018). Given their stability, universality, and capacity for selfdirected assembly, it is unsurprising that biology has repeatedly harnessed amyloids to perform functional roles, such as in bacterial cell adhesion (Chapman et al., 2002), human melanin biosynthesis (McGlinchey et al., 2009), and, intriguingly, even memory (Shorter and Lindquist, 2005;Krüttner et al., 2012). Similarly, the long-range molecular order and favorable mechanical properties of amyloids make them highly attractive for the development of nanomaterials, such as scaffolds for catalysts, templates for nanoparticles, and novel adhesives (Nguyen et al., 2014;Zhong et al., 2014;Al-Garawi et al., 2017). ...
Amyloid fibrils are a pathologically and functionally relevant state of protein folding, which is generally accessible to polypeptide chains and differs fundamentally from the globular state in terms of molecular symmetry, long-range conformational order, and supramolecular scale. Although amyloid structures are challenging to study, recent developments in techniques such as cryo-EM, solid-state NMR, and AFM have led to an explosion of information about the molecular and supramolecular organization of these assemblies. With these rapid advances, it is now possible to assess the prevalence and significance of proposed general structural features in the context of a diverse body of high-resolution models, and develop a unified view of the principles that control amyloid formation and give rise to their unique properties. Here, we show that, despite system-specific differences, there is a remarkable degree of commonality in both the structural motifs that amyloids adopt and the underlying principles responsible for them. We argue that the inherent geometric differences between amyloids and globular proteins shift the balance of stabilizing forces, predisposing amyloids to distinct molecular interaction motifs with a particular tendency for massive, lattice-like networks of mutually supporting interactions. This general property unites previously characterized structural features such as steric and polar zippers, and contributes to the long-range molecular order that gives amyloids many of their unique properties. The shared features of amyloid structures support the existence of shared structure-activity principles that explain their self-assembly, function, and pathogenesis, and instill hope in efforts to develop broad-spectrum modifiers of amyloid function and pathology.
