Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through overexpression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Overexpression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.Key words: sumoylation, mouse oocyte maturation, overexpression, Senp2, MII spindle     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important post-translational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through over-expression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Over-expression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.     

Genetic and environmental changes in SUMO homeostasis lead to nuclear mRNA retention in plants

Protein sumoylation plays an important role in plant development, flowering-time regulation, and abiotic stress response. However, the molecular role of sumoylation in these pathways is largely unknown. It was shown previously that in mutants of the inner nuclear basket nucleoporin NUA a large increase in the abundance of high-molecular weight SUMO conjugated proteins correlated with nuclear retention of bulk mRNA. Here, the connection between sumoylation and mRNA export in plants was further investigated. Both SUMO-conjugate accumulation and mRNA retention were also found in a second nucleoporin mutant that does not affect NUA, and SUMO conjugates accumulated predominantly in the nucleus. Similarly, after heat and ethanol treatment, two abiotic stress treatments known to lead to the accumulation of sumoylated proteins, nuclear mRNA was retained. To establish a causal relationship between sumoylation and mRNA export, mutations in two enzymes in the SUMO pathway were tested. Mutating either SUMO E3 ligase or SUMO isopeptidase lead to nuclear mRNA retention, indicating that both an increase and a decrease in the pool of sumoylated nuclear proteins blocks mRNA export. Together, these data show that sumoylation acts upstream of mRNA export in plants, likely through the transient sumoylation status of one or more factors involved in mRNA trafficking.     

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.     

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background

Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified.

Methodology/Principal Findings

Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci.

Conclusions/Significance

This work provides the first clear evidence for a role for SIMs in SUMO E3 activity.     

Making the connections: Autophagy and post-translational modifications in cardiomyocytes

Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein misfolding. If the protein quality control (PQC) pathways are in any way compromised or their activities diminished, intracellular aggregates can form and a proteotoxic environment is generated, which contributes to cardiac disease and heart failure. We studied the role that SUMO post-translational modification plays in a proteotoxic cardiac environment. SUMOylation can have an integral role in controlling flux through the ubiquitin-proteasome system, and expression of the SUMO (small ubiquitin-like modifier) E2 enzyme UBE2I/UBC9 improves cardiac PQC. Our data focus on using gain- and loss-of-function approaches to modify UBE2I levels and measure the effects on cardiomyocyte autophagic flux. UBE2I expression does have an impact on macroautophagy/autophagy as increased SUMOylation results in increased autophagy. We show that increased SUMOylation is cardioprotective and can decrease morbidity in proteotoxic cardiac disease.     

A genome-wide analysis of sumoylation-related biological processes and functions in human nucleus

Protein sumoylation is an important reversible post-translational modification of proteins in the nucleus, and it orchestrates a variety of the cellular processes. Genome-wide analysis of functional abundance and distribution of Small Ubiquitin-related MOdifier (SUMO) substrates may shed a light on how sumoylation is involved in nuclear biological processes and functions. Two interesting questions about sumoylation have emerged: (1) how many SUMO substrates exist in mammalian proteomes, such as human and mouse, (2) and what are their functions and how are they involved in a variety of biological processes? To address these two questions,we present an in silico genome-scale analysis for SUMO substrates in human. Based on the pattern recognition and phylogenetic conservation, we retrieved a list of 2683 potential SUMO substrates conserved in both human and mouse. Then, by functional enrichment analysis, we surveyed the over-represented GO terms and functional domains of them against the whole human proteome. Besides the consistence between our analyses and in vivo or in vitro work, the in silico predicted candidates also point to several potential roles of sumoylation, e.g., perception of sound. These potential SUMO substrates in human are of great value for further in vivo or in vitro experimental analysis.     

Correlative morphological and cytochemical observations on the nucleoli and nuclear bodies during avian oogenesis.

A correlative morphological and cytochemical study has been made of the nucleoli and nuclear bodies in the growing oocytes of the crow (Corvus splendens) and common myna (Acridotheres tristis). The nucleoli show morphological and cytochemical changes during oocyte growth, which are described in detail. In young oocytes at diplotene stage, two to six nucleoli, which are attached to condensed diplotene chromosomes, show RNA, lysine-rich histones and some non-histones, the proteins contain S-S and SH groups. In subsequent stages of oocyte growth, the cortex of nucleolus also develops arginine-rich histones whereas the medulla shows lysine-rich histones. The significance of both morphological and cytochemical changes of nucleoli has been discussed in relation to oocyte growth. Seven types of nuclear bodies are described. They are composed of proteins and carbohydrates. Their shape, size and structure vary during different stages of oocyte growth. Finally, their material is transported into the ooplasm.