Developmental Regulation and Spatiotemporal Redistribution of the Sumoylation Machinery in the Rat Central Nervous System

International audienceBACKGROUND: 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

Protein Sumoylation in Brain Development, Neuronal Morphology and Spinogenesis

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

International audienceBACKGROUND 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

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

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

International audienceMono-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

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

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Developmental regulation of the sumoylation machinery in the fractionated rat brain.

<p>Representative immunoblots of SUMO enzymes AoS1 (<b>A</b>), Ubc9 (<b>B</b>) and SENP1/6 (<b>C</b>) in nuclear and cytosolic fractions obtained from fractionated rat brains at different developmental stages. Densitometric and statistical analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033757#pone-0033757-g002" target="_blank">figure 2</a> legend and graphic representations show means ± s.e.m. of five independent experiments. (<b>A</b>) **p<0.001 compared with other age points and *p<0.05 compared with adult. (<b>B</b>) **p<0.01 compared with adult and *p<0.001 compared with adult. (<b>C</b>) **p<0.01 compared with adult and *p<0.001 compared with other age points.</p

Presynaptic redistribution of the sumoylation machinery during neuronal maturation.

<p>Confocal images show the colocalisation (yellow) between the presynaptic marker Bassoon in green and in red, the sumoylation enzymes AoS1 and Ubc9 (<b>A</b>) or the desumoylases SENP1 and SENP6 (<b>B</b>) in 10 and 20 DIV cultured rat hippocampal neurons. Enlargement of hatched areas are also depicted. Scale bars, 20 µm. Quantification of presynaptic colocalisation was performed using the ImageJ software as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033757#s4" target="_blank">methods</a>. Histograms represent the relative presynaptic intensity of the sumoylation machinery and each value is the mean ± s.e.m. measured from 40 cells in four independent experiments. (<b>A</b>) Student's <i>t</i>-tests, *p<0.0001; n.s., not significant. (<b>B</b>) Student's <i>t</i>-tests, *p<0.05 and **p<0.001.</p

Developmental regulation of the sumoylation pathway in the rat brain.

<p>Representative developmental expression profiles of SUMO1- and SUMO2/3-modified protein substrates and sumoylation (AoS1, Ubc9) and desumoylation (SENP1 and SENP6) enzymes. Whole rat brain homogenates at different ages, ranging from the embryonic day E9 to the post-natal day P14 and the adult (Ad) stage, were prepared in the presence of NEM to protect proteins from desumoylation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033757#s4" target="_blank">Method</a> section. Lower panel shows immunoblot of standard ß-actin loading control.</p

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