This is the second edition of a very well received book that details how the sumoylation system functions and how it modulates numerous cellular activities. SUMO is a post-translational modifier in the ubiquitin super-family that has gained recognition over the last twenty years as an essential and prevalent regulatory molecule. Individual chapters explore the biochemistry, molecular biology, and cell biology of the sumoylation system and its substrate proteins. The book is divided into three themed parts: Molecular Functions (I), Cell Growth Regulation (II), and Diseases (III). Parts I and II focus on the contribution of sumoylation to cellular activities in both the nuclear and cytoplasmic compartments. The nuclear activities covered include nucleic acid metabolism (both RNA and DNA), chromosome structure and replication, and nucleocytoplasmic transport. Cytoplasmic processes presented include regulation of membrane ion channels, general metabolism, and apoptotic signalling. Topics in Part III include the role of sumoylation in developmental abnormalities (craniofacial and cardiovascular), diabetes, neurodegenerative diseases, cancer, and infections with viruses and bacteria. Each of the corresponding chapter authors is an active researcher who has made significant contributions to understanding sumoylation. This second edition provides updates and revisions to most of the original chapters plus adds six new chapters to address important developing areas of sumoylation research. This volume is intended for a scientific audience from undergraduates to independent researchers. The content will serve as both a solid introduction for the novice reader and an in depth treatment for the advanced scholar.

This volume explores various methodologies to study biochemical, molecular, and cellular biology aspects of some processes regulated by protein SUMOylation. SUMO: Methods and Protocols is organized into four parts, and starts with an historical overview on protein SUMOylation and a presentation of the methods included in the book. The first part also includes a review on chromatin regulation by dynamic SUMO modifications. The second part focuses on in vitro techniques, including biochemical methods to study mechanistic aspects of protein SUMOylation. The third part includes protocols to be used with cell cultures, which often are the first approaches used in most laboratories. The final part includes methodologies adapted for the analysis in vivo using distinct model organisms. Written in the highly successful Methods in Molecular Biology series format, chapters include a brief introduction to the subject, a list of necessary materials and reagents, a step-by-step reproducible laboratory protocol ending with a Notes section on troubleshooting tips, and tips and strategies to avoid known pitfalls. Unique and cutting-edge, SUMO: Methods and Protocols provides a comprehensive source of protocols for specialists and researchers not familiar with this vital system.

SUMOylation, a post-translational modification with SUMO proteins covalently conjugated to a variety of proteins, regulates a range of cellular processes, including cell proliferation and maintenance of genome stability. In this study, we investigated how SUMO-1 functions as a chromatin mark on the human genome during cell cycle progression by ChIP-seq approach. Surprisingly, despite the known repressive role of SUMOylation on histones, we found that SUMO-1 localizes to the promoters of constitutively active genes involved in protein translation and proliferation during interphase. For example, ribosomal protein genes; and SUMO-1 marks on these promoters were absent during mitosis. In addition, SUMO-1 association on the promoters recruits RNAPII, and depletion of SUMO-1 leads to down regulation of those ribosomal protein genes, suggesting a positive role of SUMO-1 in gene activation. To further elucidate how SUMOylation regulates transcription process related to protein synthesis, we identified that SUMO-1 marks the promoters via the Scaffold Associated Factor B (SAFB) protein. The results showed that SAFB is SUMOylated, and depletion of SAFB caused the decrease of SUMO-1 marks on the promoters of those housekeeping genes transcribed by RNAPII. In addition, depletion of SAFB decreased the splicing of the mRNAs and disrupted the organization of Cajal body, which is important for snRNP and snoRNP biogenesis. All these findings suggested that SUMOylation plays an important role in the regulatory process for transcription initiation and splicing of mRNA of ribosomal protein genes.

ABSTRACT: Covalent attachment of the Small Ubiquitin-like Modifier (SUMO) to proteins in eukaryotic cells has been demonstrated to be an important step in the regulation of a wide variety of cellular processes. Such processes include transcriptional regulation, mediating DNA/protein interaction, and increasing protein stability by interfering with the ubiquitin conjugation pathway in some cases. While much work has been done to characterize the SUMO pathway in yeast, insect, and mammalian systems, relatively little is known about the roles that SUMO conjugation plays in plants. In this study, I make use of the Populus model system to investigate the gene structures of the various family members of the SUMO pathway in plants, as well as demonstrate that the pathway is active in a number of tissue types. Additionally, we show that the SUMOylation is inducible through acute stress treatments such as desiccation, heat shock, and peroxide application. To further our understanding of the SUMOylation process, I have set out to identify SUMO-conjugated proteins from heat shock treated tissues through a multi-step purification process.

SUMO modification is involved in several cellular processes such as signal transduction, cell division, and regulation of protein subcellular localization, specifically including Ras signaling and protein nuclear import/export. Previous studies have demonstrated that 14-3-3[zeta], which is thought to have functions in both the nucleus and cytoplasm, is a SUMOylated protein and is required for Ras signaling. I have therefore sought to determine whether or not the SUMOylation of 14-3-3[zeta] promotes or inhibits its localization to the nucleus. Subcellular fractionation experiments fail to reveal a detectable change in the overall ratio of nuclear to cytoplasmic 14-3-3[zeta] upon knockdown of SUMO by RNAi in cultured Drosophila cells. However, immunofluorescence studies suggest that nuclear localization of this protein may increase in a subset of the SUMO knockdown cells.

Most proteins undergo post-translational modifications altering physical and chemical properties, folding, conformation distribution, stability, activity and function. Ubiquitin and SUMOs are related small proteins that are members of the large ubiquitin superfamily of post-translational modifiers. Written by highly respected leaders in their fields under the expert guidance of the editor, this volume covers the principles of ubiquitination and SUMOylation, presents detailed reviews of current and emerging concepts and highlights new advances in all areas of SUMOylation and ubiquitination. Topics of note include: the ubiquitin superfamily, the ubiquitin toolbox, onco viral exploitation of the SUMO system, small molecule modulators of desumoylation, mass spectrometry, global proteomic profiling of SUMO and ubiquitin, biotin-based approaches, genetic screening, SUMOylation networks in humans, targets for ubiquitin ligases, regulation of p53, protein homeostasis, miRNAs, DNA replication, DNA damage response, telomere biology, intracellular trafficking, regulation of angiogenesis, brain ischemia, autophagy, assembly and activity, antiviral defense, HIV infection, amyloid and amyloid-like proteins, plant immunity. This comprehensive and up-to-date book is the definitive reference volume on all aspects of SUMOylation and ubiquitination and is an essential acquisition for anyone involved in this area of biology.

Cells possess a wealth of posttranscriptional control mechanisms that impact on every conceivable aspect of the life of an mRNA. These processes are intimately intertwined in an almost baroque manner, where promoter context influences the recruitment of splicing factors, where the majority of pre-mRNAs undergo alternative splicing, and where proteins deposited during nuclear processing impact distal cytoplasmic processing, translation, and decay. If there is a unifying theme to mRNA Processing and Metabolism: Methods and Protocols, it is that mRNA processing and metabolism are integrated processes. Many of the techniques used to study mRNA have been described in a previous volume of this series (RNA–Protein Interaction Protocols, Susan Haynes, ed.) and specialized methods journals. In selecting topics for mRNA Processing and Metabolism: Methods and Protocols, I sought input on new and novel techniques and approaches that build on this foundation using technological advances in microscopy, whole genome sequencing, microarrays, mass spectrometry, fluorescent detection methodologies, and RNA interference. I have tried not to bias this book toward any single model organism, and approaches described in the various chapters use yeast, Drosophila, Xenopus, mice, plants, and cultured mammalian cells.