Combining affinity purification and mass spectrometry to define the network of the nuclear proteins interacting with the N-terminal region of FMRP.

Fragile X-Syndrome (FXS) represents the most common inherited form of intellectual disability and the leading monogenic cause of Autism Spectrum Disorders. In most cases, this disease results from the absence of expression of the protein FMRP encoded by the FMR1 gene (Fragile X messenger ribonucleoprotein 1). FMRP is mainly defined as a cytoplasmic RNA-binding protein regulating the local translation of thousands of target mRNAs. Interestingly, FMRP is also able to shuttle between the nucleus and the cytoplasm. However, to date, its roles in the nucleus of mammalian neurons are just emerging. To broaden our insight into the contribution of nuclear FMRP in mammalian neuronal physiology, we identified here a nuclear interactome of the protein by combining subcellular fractionation of rat forebrains with pull- down affinity purification and mass spectrometry analysis. By this approach, we listed 55 candidate nuclear partners. This interactome includes known nuclear FMRP-binding proteins as Adar or Rbm14 as well as several novel candidates, notably Ddx41, Poldip3, or Hnrnpa3 that we further validated by target-specific approaches. Through our approach, we identified factors involved in different steps of mRNA biogenesis, as transcription, splicing, editing or nuclear export, revealing a potential central regulatory function of FMRP in the biogenesis of its target mRNAs. Therefore, our work considerably enlarges the nuclear proteins interaction network of FMRP in mammalian neurons and lays the basis for exciting future mechanistic studies deepening the roles of nuclear FMRP in neuronal physiology and the etiology of the FXS.

Proteomics datasets of developing rat brain: Synaptic proteome and SUMO2/3-ylome.

During brain development, synapses undergo structural rearrangements and functional changes mediated by many molecular processes including post-translational modifications by the Small Ubiquitin-like MOdifier (SUMO). To get an overview of the endogenous SUMO-modified proteins in the developing rat brain synapses, our first aim was to characterize the synaptic proteome from rat at 14 postnatal days (PND14), a period that combines intense synaptogenesis, neurotransmission and high levels of SUMO2/3-ylation. In this purpose, we isolated the synaptosomal fraction by differential centrifugation on sucrose percoll gradient and characterized the synaptosomal proteome by nanoLC-MS/MS. Our second aim was to provide a comprehensive list of the SUMO2/3-modified protein in this compartment. We thus performed an enrichment in SUMO2/3-ylated proteins from the synaptosomal fraction by denaturing immunoprecipitation using specific anti-SUMO2/3 antibodies prior to proteomics analysis. The information presented in this article complement the publication "Proteomic Identification of an Endogenous Synaptic SUMOylome in the Developing Rat Brain" [1], by focusing on the characterization of the synaptic proteome of PND14 rat brain. Altogether, these data can inform future experiments focused on studying the functional consequences of synaptic SUMOylation regarding synapses structure and function. In addition, they can provide the basis for future mechanistic studies investigating brain pathologies involving altered SUMOylation levels.

Proteomic Identification of an Endogenous Synaptic SUMOylome in the Developing Rat Brain.

Synapses are highly specialized structures that interconnect neurons to form functional networks dedicated to neuronal communication. During brain development, synapses undergo activity-dependent rearrangements leading to both structural and functional changes. Many molecular processes are involved in this regulation, including post-translational modifications by the Small Ubiquitin-like MOdifier SUMO. To get a wider view of the panel of endogenous synaptic SUMO-modified proteins in the mammalian brain, we combined subcellular fractionation of rat brains at the post-natal day 14 with denaturing immunoprecipitation using SUMO2/3 antibodies and tandem mass spectrometry analysis. Our screening identified 803 candidate SUMO2/3 targets, which represents about 18% of the synaptic proteome. Our dataset includes neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins as well as vesicular trafficking and cytoskeleton-associated proteins, defining SUMO2/3 as a central regulator of the synaptic organization and function.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice.

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

The synaptic balance between sumoylation and desumoylation is maintained by the activation of metabotropic mGlu5 receptors.

Sumoylation is a reversible post-translational modification essential to the modulation of neuronal function, including neurotransmitter release and synaptic plasticity. A tightly regulated equilibrium between the sumoylation and desumoylation processes is critical to the brain function and its disruption has been associated with several neurological disorders. This sumoylation/desumoylation balance is governed by the activity of the sole SUMO-conjugating enzyme Ubc9 and a group of desumoylases called SENPs, respectively. We previously demonstrated that the activation of type 5 metabotropic glutamate receptors (mGlu5R) triggers the transient trapping of Ubc9 in dendritic spines, leading to a rapid increase in the overall synaptic sumoylation. However, the mechanisms balancing this increased synaptic sumoylation are still not known. Here, we examined the diffusion properties of the SENP1 enzyme using a combination of advanced biochemical approaches and restricted photobleaching/photoconversion of individual hippocampal spines. We demonstrated that the activation of mGlu5R leads to a time-dependent decrease in the exit rate of SENP1 from dendritic spines. The resulting post-synaptic accumulation of SENP1 restores synaptic sumoylation to initial levels. Altogether, our findings reveal the mGlu5R system as a central activity-dependent mechanism to maintaining the homeostasis of sumoylation at the mammalian synapse.

Sumoylation regulates FMRP-mediated dendritic spine elimination and maturation.

Fragile X syndrome (FXS) is the most frequent inherited cause of intellectual disability and the best-studied monogenic cause of autism. FXS results from the functional absence of the fragile X mental retardation protein (FMRP) leading to abnormal pruning and consequently to synaptic communication defects. Here we show that FMRP is a substrate of the small ubiquitin-like modifier (SUMO) pathway in the brain and identify its active SUMO sites. We unravel the functional consequences of FMRP sumoylation in neurons by combining molecular replacement strategy, biochemical reconstitution assays with advanced live-cell imaging. We first demonstrate that FMRP sumoylation is promoted by activation of metabotropic glutamate receptors. We then show that this increase in sumoylation controls the homomerization of FMRP within dendritic mRNA granules which, in turn, regulates spine elimination and maturation. Altogether, our findings reveal the sumoylation of FMRP as a critical activity-dependent regulatory mechanism of FMRP-mediated neuronal function.

Physiological and pathophysiological control of synaptic GluN2B-NMDA receptors by the C-terminal domain of amyloid precursor protein.

The amyloid precursor protein (APP) harbors physiological roles at synapses and is central to Alzheimer's disease (AD) pathogenesis. Evidence suggests that APP intracellular domain (AICD) could regulate synapse function, but the underlying molecular mechanisms remain unknown. We addressed AICD actions at synapses, per se, combining in vivo AICD expression, ex vivo AICD delivery or APP knock-down by in utero electroporation of shRNAs with whole-cell electrophysiology. We report a critical physiological role of AICD in controlling GluN2B-containing NMDA receptors (NMDARs) at immature excitatory synapses, via a transcription-dependent mechanism. We further show that AICD increase in mature neurons, as reported in AD, alters synaptic NMDAR composition to an immature-like GluN2B-rich profile. This disrupts synaptic signal integration, via over-activation of SK channels, and synapse plasticity, phenotypes rescued by GluN2B antagonism. We provide a new physiological role for AICD, which becomes pathological upon AICD increase in mature neurons. Thus, AICD could contribute to AD synaptic failure.

mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines.

Sumoylation plays important roles in the modulation of protein function, neurotransmission and plasticity, but the mechanisms regulating this post-translational system in neurons remain largely unknown. Here we demonstrate that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the sumoylation pathway, is regulated by synaptic activity. We use restricted photobleaching/photoconversion of individual hippocampal spines to measure the diffusion properties of Ubc9 and show that it is regulated through an mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABAA receptor antagonist or directly activating mGlu5R increases the synaptic residency time of Ubc9 via a Gαq/PLC/Ca(2+)/PKC cascade. This activation promotes a transient synaptic trapping of Ubc9 through a PKC phosphorylation-dependent increase of Ubc9 recognition to phosphorylated substrates and consequently leads to the modulation of synaptic sumoylation. Our data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory processes and provide a mechanism for the dynamic changes in sumoylation occurring during synaptic transmission.

Protein sumoylation in brain development, neuronal morphology and spinogenesis.

Small ubiquitin-like modifiers (SUMOs) are polypeptides resembling ubiquitin that are covalently attached to specific lysine residue of target proteins through a specific enzymatic pathway. Sumoylation is now seen as a key posttranslational modification involved in many biological processes, but little is known about how this highly dynamic protein modification is regulated in the brain. Disruption of the sumoylation enzymatic pathway during the embryonic development leads to lethality revealing a pivotal role for this protein modification during development. The main aim of this review is to briefly describe the SUMO pathway and give an overview of the sumoylation regulations occurring in brain development, neuronal morphology and synapse formation.

Activity-dependent regulation of the sumoylation machinery in rat hippocampal neurons.

BACKGROUND INFORMATION: Sumoylation is a key post-translational modification by which the Small Ubiquitin-like MOdifier (SUMO) polypeptide is covalently attached to specific lysine residues of substrate proteins through a specific enzymatic pathway. Although sumoylation participates in the regulation of nuclear homeostasis, the sumoylation machinery is also expressed outside of the nucleus where little is still known regarding its non-nuclear functions, particularly in the Central Nervous System (CNS). We recently reported that the sumoylation process is developmentally regulated in the rat CNS. RESULTS: Here, we demonstrate that there is an activity-dependent redistribution of endogenous sumoylation enzymes in hippocampal neurons. By performing biochemical and immunocytochemical experiments on primary cultures of rat hippocampal neurons, we show that sumoylation and desumoylation enzymes are differentially redistributed in and out of synapses upon neuronal stimulation. This enzymatic redistribution in response to a neuronal depolarisation results in the transient decrease of sumoylated protein substrates at synapses. CONCLUSIONS: Taken together, our data identify an activity-dependent regulation of the sumoylation machinery in neurons that directly impacts on synaptic sumoylation levels. This process may provide a mechanism for neurons to adapt their physiological responses to changes occurring during neuronal activation.

Developmental regulation and spatiotemporal redistribution of the sumoylation machinery in the rat central nervous system.

BACKGROUND: Small Ubiquitin-like MOdifier protein (SUMO) is a key regulator of nuclear functions but little is known regarding the role of the post-translational modification sumoylation outside of the nucleus, particularly in the Central Nervous System (CNS). METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the expression levels of SUMO-modified substrates as well as the components of the sumoylation machinery are temporally and spatially regulated in the developing rat brain. Interestingly, while the overall sumoylation is decreasing during brain development, there are progressively more SUMO substrates localized at synapses. This increase is correlated with a differential redistribution of the sumoylation machinery into dendritic spines during neuronal maturation. CONCLUSIONS/SIGNIFICANCE: Overall, our data clearly demonstrate that the sumoylation process is developmentally regulated in the brain with high levels of nuclear sumoylation early in the development suggesting a role for this post-translational modification during the synaptogenesis period and a redistribution of the SUMO system towards dendritic spines at a later developmental stage to modulate synaptic protein function.

H2B ubiquitylation controls the formation of export-competent mRNP.

Histone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs. While H2B ubiquitylation controls the recruitment of the different Mex67 adaptors to mRNPs, the effect of Swd2 ubiquitylation is restricted to Yra1 and Nab2, which, in turn, controls poly(A) tail length. Modification of H2B thus participates in the crosstalk between cotranscriptional events and assembly of mRNPs linking nuclear processing and mRNA export.

Competition for XPO5 binding between Dicer mRNA, pre-miRNA and viral RNA regulates human Dicer levels.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA-exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.

Ubiquitin-mediated mRNP dynamics and surveillance prior to budding yeast mRNA export.

The evolutionarily conserved mRNA export receptor Mex67/NXF1 associates with mRNAs through its adaptor, Yra1/REF, allowing mRNA ribonucleoprotein (mRNP) exit through nuclear pores. However, alternate adaptors should exist, since Yra1 is dispensable for mRNA export in Drosophila and Caenorhabditis elegans. Here we report that Mex67 interacts directly with Nab2, an essential shuttling mRNA-binding protein required for export. We further show that Yra1 enhances the interaction between Nab2 and Mex67, and becomes dispensable in cells overexpressing Nab2 or Mex67. These observations appoint Nab2 as a potential adaptor for Mex67, and define Yra1/REF as a cofactor stabilizing the adaptor-receptor interaction. Importantly, Yra1 ubiquitination by the E3 ligase Tom1 promotes its dissociation from mRNP before export. Finally, loss of perinuclear Mlp proteins suppresses the growth defects of Tom1 and Yra1 ubiquitination mutants, suggesting that Tom1-mediated dissociation of Yra1 from Nab2-bound mRNAs is part of a surveillance mechanism at the pore, ensuring export of mature mRNPs only.

Ubiquitylation of the COMPASS component Swd2 links H2B ubiquitylation to H3K4 trimethylation.

Mono-ubiquitylation of histone H2B correlates with transcriptional activation and is required for di- and trimethylation at Lys 4 on the histone H3 tail (H3K4) by the SET1/COMPASS methyltransferase complex through a poorly characterized trans-tail pathway. Here we show that mono-ubiquitylation of histone H2B promotes ubiquitylation at Lys 68 and Lys 69 of Swd2, the essential component of SET1/COMPASS in Saccharomyces cerevisiae. We found that Rad6/Bre1 ubiquitylation enzymes responsible for H2B ubiquitylation also participate directly in Swd2 modification. Preventing Swd2 or H2B ubiquitylation did not affect Set1 stability, interaction of Swd2 with Set1 or the ability of Swd2 to interact with chromatin. However, we found that mutation of Lys 68 and Lys 69 of Swd2 markedly reduced trimethylation, and to a lesser extent dimethylation, of H3K4 at the 5'-end of transcribing genes without affecting monomethylation. This effect results from the ability of Swd2 ubiquitylation to control recruitment of Spp1, a COMPASS subunit necessary for trimethylation. Our results further indicate that Swd2 is a major H3-binding component of COMPASS. Swd2 thus represents a key factor that mediates crosstalk between H2B ubiquitylation and H3K4 trimethylation on chromatin.

Coordination of Hpr1 and ubiquitin binding by the UBA domain of the mRNA export factor Mex67.

The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps in coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here, we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but it decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind polyubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes, and it also shows an mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for polyubiquitin, and prevents its binding to nonspecific substrates.

Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription.

The mRNA nuclear export receptor Mex67/Mtr2 is recruited to mRNAs through RNA-binding adaptors, including components of the THO/TREX complex that couple transcription to mRNA export. Here we show that the ubiquitin-associated (UBA) domain of Mex67 is not only required for proper nuclear export of mRNA but also contributes to recruitment of Mex67 to transcribing genes. Our results reveal that the UBA domain of Mex67 directly interacts with polyubiquitin chains and with Hpr1, a component of the THO/TREX complex, which is regulated by ubiquitylation in a transcription-dependent manner. This interaction transiently protects Hpr1 from ubiquitin/proteasome-mediated degradation and thereby coordinates recruitment of the mRNA export machinery with transcription and early messenger ribonucleoproteins assembly.

The mRNA nuclear export factor Hpr1 is regulated by Rsp5-mediated ubiquitylation.

Ubiquitin conjugation and in particular two distinct HECT ubiquitin ligases, Rsp5p and Tom1p, have been shown to participate in the regulation of mRNA export in Saccharomyces cerevisiae. The identification of the ubiquitin ligase substrates represents a major challenge in understanding how this modification may modulate mRNA export. Here, we identified Hpr1p, a member of the THO/TREX (transcription/export) complex that couples mRNA transcription to nuclear export as a target of the ubiquitin-proteasome pathway. Hpr1p degradation is enhanced at high temperature and appears linked to on-going RNA-polymeraseII-mediated transcription. Interestingly, the stability of the other THO complex components is not affected under these conditions indicating that Hpr1p turnover could control the formation of the THO/TREX complex and consequently mRNA export. Using in vivo and in vitro approaches we demonstrate that Rsp5p is responsible for the ubiquitylation of Hpr1p that also involves the ubiquitin-conjugating enzyme Ubc4p. Thus, Hpr1p represents the first nuclear export factor regulated by ubiquitylation, strongly suggesting that this post-translational modification participates in the coordination of transcription and mRNA export processes.

Minihelix-containing RNAs mediate exportin-5-dependent nuclear export of the double-stranded RNA-binding protein ILF3.

The karyopherin-related nuclear transport factor exportin-5 preferentially recognizes and transports RNAs containing minihelix motif, a structural cis-acting export element that comprises a double-stranded stem (>14 nucleotides) with a base-paired 5' end and a 3-8-nucleotide protruding 3' end. This structural motif is present in various small cellular and viral polymerase III transcripts such as the adenovirus VA1 RNA (VA1). Here we show that the double-stranded RNA-binding protein, ILF3 (interleukin enhancer binding factor 3) preferentially binds minihelix motif. Gel retardation assays and glutathione S-transferase pull-down experiments revealed that ILF3, exportin-5, RanGTP, and VA1 RNA assembled in a quaternary complex in which the RNA moiety bridges the interaction between ILF3 and exportin-5. Formation of this complex is facilitated by the ability of both exportin-5 and ILF3 to mutually increase their apparent affinity for VA1 RNA. Using microinjection in the nucleus of HeLa cells and transfection experiments, we show here that formation of the cooperative RanGTP-dependent RNA/ILF3/exportin-5 complex promotes the co-transport of VA1 and ILF3 from the nucleus to the cytoplasm. Exportin-5 thus appears as the first example of a nuclear export receptor that mediates RNA export but also promotes transport of proteinaceous cargo through appropriate and specific RNA adaptors.