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TAP purification of SUMO-2 from cultured human cells. (A) Schematic representation of the TAP–SUMO-2 construct with which HeLa cells were stably transfected. The TAP tag consists of a protein A domain separated from a calmodulin-binding protein (CBP) domain by the tobacco etch virus (TEV) protease site. This was N-terminally tagged to SUMO-2 (amino acid residues 1 to 92) (NCBI Entrez protein CAG46970), which can be conjugated directly to target proteins through a covalent bond. (B) An antibody against SUMO-2 was used for the analysis of Western blots of crude cell lysates from TAP–SUMO-2-containing HeLa cells under normal conditions (37°C) and after heat shock for 30 min (43°C). This Western blot is representative of five separate experiments. (C) Silver-stained SDS-PAGE gel showing TAP purification products from HeLa cells containing TAP alone or TAP–SUMO-2. This stained gel is representative of six separate experiments.
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... identify the cellular targets of stress-induced SUMOylation, a quantita- tive proteomic approach was taken. We generated a HeLa cell line that con- tained SUMO-2 fused to a tandem affinity protein (TAP) tag (10) ( Fig. 2A), which was similar in its abundance and its response to heat shock to that of endogenous SUMO-2 (Fig. 2B). Comparison of the products purified under initially denaturing conditions from HeLa cells containing only the TAP tag with those from cells containing TAP-SUMO-2 confirmed the high strin- gency of this method (Fig. 2C) and, hence, ...
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... identify the cellular targets of stress-induced SUMOylation, a quantita- tive proteomic approach was taken. We generated a HeLa cell line that con- tained SUMO-2 fused to a tandem affinity protein (TAP) tag (10) ( Fig. 2A), which was similar in its abundance and its response to heat shock to that of endogenous SUMO-2 (Fig. 2B). Comparison of the products purified under initially denaturing conditions from HeLa cells containing only the TAP tag with those from cells containing TAP-SUMO-2 confirmed the high strin- gency of this method (Fig. 2C) and, hence, its suitability for proteomic anal- ysis. For quantitation of the proteome, we used the stable isotope ...
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... (TAP) tag (10) ( Fig. 2A), which was similar in its abundance and its response to heat shock to that of endogenous SUMO-2 (Fig. 2B). Comparison of the products purified under initially denaturing conditions from HeLa cells containing only the TAP tag with those from cells containing TAP-SUMO-2 confirmed the high strin- gency of this method (Fig. 2C) and, hence, its suitability for proteomic anal- ysis. For quantitation of the proteome, we used the stable isotope labeling of amino acids in cell culture (SILAC) technique, in which isotopically unique forms of amino acids are used to measure the relative abundance of proteins in cells grown under different conditions (11). For our ...
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... blotting analysis showed that during heat shock, the number of proteins modified by SUMO-2 increased and the amount of free SUMO-2 was depleted compared to that in cells in the absence of heat shock (7) (Fig. 2B). Although analysis of the tsMap confirmed that heat shock induced the SUMOylation of a substantial number of proteins ( Fig. 3B, vertical histogram), it is also clear that this occurred concomitantly with the deSUMOylation of a different subset of proteins, which was detectably modified under basal conditions. This was apparent as a ...
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... better understand the temporal dynamics of the SUMOylation response to heat shock, we applied the same quantitative approach to monitor the system-wide changes in SUMOylation of proteins both in response to heat shock and after a 2-hour heat shock recovery period ( fig. S2 and Fig. ...
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... the global increase in SUMO conjugation in response to heat shock observed by Western blotting experiments was rapid, with almost all free SUMO proteins being conjugated into high molecular weight adducts after 5 min, deconjugation during the recovery period was much slower ( fig. S2). System-wide analysis of the SUMO-2 substrate proteome by tsMap ( fig. S3B) revealed a general trend whereby proteins SUMOylated upon heat shock became deconjugated during the recovery period [for example, the glucocorticoid receptor (GR) (Fig. 4B)], but those proteins that were deconjugated during the heat shock response mostly did ...
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... findings raise the important question of how the stress signal is transduced to lead to altered SUMOylation. Because modification by SUMO proteins was already almost maximal 5 min after exposure to heat shock ( fig. S2), this suggests that transmission of the message from the sensor of increased temperature to the enzymes that influence SUMO modification must be fairly direct. This might involve either increasing the activity of the conjugation machinery or decreasing the activity of the SUMO-specific proteases (SENPs) that are responsible for ...
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... Fig. S1. Double quantitative filtering with tsMaps. Fig. S2. Effect of recovery from heat shock on SUMO-2 conjugates in HeLa cells. . Filtering putative substrates of SUMO-2 from SILAC experiment 2. Fig. S4. Comparison between crude and TAP-purified cell lysates for changes in protein abundance after heat shock and recovery. Fig. S5. Comparisons between the numbers of identified proteins in ...
Citations
... Although our previous work as well as high-throughput studies have identified EXOSC10 as a target of SUMOylation [31,[46][47][48], the role of this modification in the EXOSC10 function or the cellular response to hypoxia has not been previously addressed. Here, we report the identification of the enzymes responsible for EXOSC10 SUMOylation, and deSUMOylation, and describe how these processes and EXOSC10 function respond to low oxygen conditions. ...
... Previously, we and others [33,49,53,61] identified EXOSC10 as a SUMOylated protein in large-scale proteomic screens. Interestingly, heat shock modulates SUMOylation of EXOSC10 [47], while cooling-induced modification of EXOSC10 by SUMO was shown to reduce its protein levels [62], suggesting that regulation of EXOSC10 SUMOylation may be a common strategy for fine-tuning responses to different stress conditions. However, until recently, the enzymes involved in EXOSC10 SUMOylation had not been characterized. ...
Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications; however, these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in an HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme-mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00018-023-05035-9.
... Conjugation of ubiquitin to eIF5A has been shown to modulate its stability and proteasome-mediated degradation [21], and while acetylation inactivates the protein [22], hypusine modification is essential for eIF5A-dependent formation of SGs, eukaryotic cell proliferation [7,16,[23][24][25], and Saccharomyces cerevisiae viability [26]. Several proteomic studies have also pointed to eIF5A as a putative stressinduced SUMOylation target [27][28][29][30]. However, whether eIF5A is modified by small ubiquitin-like modifier (SUMO) and what the relevance of this modification is for eIF5A activities are still unknown. ...
... Proteomic data pointed to eIF5A protein as a potential SUMO substrate [27][28][29]. ...
... In silico analysis of the eIF5A1 amino acid sequence using the web servers GPS-SUMO [53] and JASSA [54] as well as previously reported proteomic data [27][28][29] suggested that different lysine residues in eIF5A1 such as K27, K34, K39, K67, K68, K85, and K126 can work as SUMO acceptors. Evaluation of single mutants of eIF5A1 in each of these lysine residues did not reveal a reduction in the eIF5A1 SUMOylation ( Fig. 2A). ...
Background
The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown.
Methods
SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6–SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1–GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student’s t -test, or one-way or two-way analysis of variance (ANOVA).
Results
We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells.
Conclusions
SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.
Graphical Abstract
... SUMOylation regulates these cellular processes by altering the target protein's activity, localization, stability, or interacting ability with binding partners (3). SUMOylation has long been associated with stress responses, integrating a diverse array of cellular stress signals that trigger rapid changes in global protein SUMOylation (4)(5)(6)(7). Dysregulation of cellular SUMOylation has been implicated in the development of human diseases such as atherosclerosis, autoimmune diseases, cancer, diabetes, heart failure, and neurological disorders (8). ...
... When the human EPAC1b sequence was analyzed using a SUMOylation prediction algorithm (19), several putative SCMs, as well as a SUMO-interacting motif (SIM) (20,21), were identified (Table S1). To test if EPAC1 is SUMOylated in cells, we subjected cells to heat shock treatment, known to induce robust cellular SUMOylation (6). We subsequently probed the status of EPAC1, endogenously in Human Umbilical Vein Endothelial Cells (HUVECs) or ectopically expressed in HEK293 cells, which expresses a very low level of EPAC1 (9), by immunoblotting using a monoclonal EPAC1 antibody, 5D3. ...
Exchange protein directly activated by cAMP (EPAC1) mediates the intracellular functions of a critical stress-response second messenger, cAMP. Herein, we report that EPAC1 is a cellular substrate of protein SUMOylation, a prevalent stress-response posttranslational modification. Site-specific mapping of SUMOylation by mass spectrometer leads to identifying K561 as a primary SUMOylation site in EPAC1. Sequence and site-directed mutagenesis analyses reveal a functional SUMO-interacting motif required for cellular SUMOylation of EPAC1. SUMO modification of EPAC1 mediates its heat shock-induced Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.
... Despite major technical challenges in site-specific characterization of protein SUMOylation, largely due to the low stoichiometry of SUMO-conjugation, a large number of SUMOylated proteins have been identified in cultured human cells using various mass spectrometry (MS)-based proteomics approaches (Becker et al., 2013;Golebiowski et al., 2009;Hendriks et al., 2014;Hendriks et al., 2017;Hendriks and Vertegaal, 2016;Lamoliatte et al., 2014;Lumpkin et al., 2017;Tammsalu et al., 2014). A comprehensive mapping of the human SUMO proteome has led to the identification of more than 40,000 SUMO modification sites in 6,747 human proteins (Hendriks et al., 2017), which are approximately one-third of the entire human proteome. ...
... (Bergink and Jentsch, 2009;Vertegaal, 2022). Additionally, unlike protein phosphorylation, which is frequently triggered by well-defined signals, such as growth factors or second messengers, the biological stimuli that regulate SUMOylation are poorly understood: SUMOylation is regulated by an array of diverse cellular stress signals such as heat shock or oxidative stress that are more global and less specific in nature (Golebiowski et al., 2009;Saitoh and Hinchey, 2000;Yang et al., 2012;Zhou et al., 2004). ...
Protein SUMOylation is a major post-translational modification important for maintaining cellular homeostasis. SUMOylation has long been associated with stress responses as a diverse array of cellular stress signals are known to trigger rapid alternations in global protein SUMOylation. In addition, while there are large families of ubiquitination enzymes, all SUMOs are conjugated by a set of enzymatic machinery comprising one heterodimeric SUMO-activating enzyme, a single SUMO-conjugating enzyme, and a small number of SUMO protein ligases and SUMO-specific proteases. How a few SUMOylation enzymes specifically modify thousands of functional targets in response to diverse cellular stresses remains an enigma. Here, we review recent progress toward understanding the mechanisms of SUMO regulation, particularly, the potential roles of liquid-liquid phase separation/biomolecular condensates in the regulation of cellular SUMOylation during cellular stresses. In addition, we discuss the role of protein SUMOylation in pathogenesis and the development of novel therapeutics targeting SUMOylation. Significance Statement Protein SUMOylation is one of the most prevalent post-translational modifications and plays an important role in maintaining cellular homeostasis in response to stresses. Protein SUMOylation has been implicated in human pathogenesis such as cancer, cardiovascular diseases, neurodegeneration, and infection. After more than a quarter century of extensive research, intriguing enigmas remain regarding the mechanism of cellular SUMOylation regulation and the therapeutic potential of targeting SUMOylation.
... SUMO-2 and SUMO-3 are required for cells to survive heat shock. In summary, SUMO is polymerized into polySUMO chains in response to heat shock and is redistributed to a regulated wide range of protein functions, including folding, transcription, translation, cell cycle regulation, DNA replication and apoptosis (99). SUMO-1 localization on chromatin is dynamic throughout the cell cycle by using chromatin affinity purification coupled with nextgeneration sequencing, which is consistent with the reversible nature of SUMOylation (100). ...
SUMOylation is a reversible post-translational modification which has emerged as a crucial molecular regulatory mechanism, involved in the regulation of DNA damage repair, immune responses, carcinogenesis, cell cycle progression and apoptosis. Four SUMO isoforms have been identified, which are SUMO1, SUMO2/3 and SUMO4. The small ubiquitin-like modifier (SUMO) pathway is conserved in all eukaryotes and plays pivotal roles in the regulation of gene expression, cellular signaling and the maintenance of genomic integrity. The SUMO catalytic cycle includes maturation, activation, conjugation, ligation and de-modification. The dysregulation of the SUMO system is associated with a number of diseases, particularly cancer. SUMOylation is widely involved in carci-nogenesis, DNA damage response, cancer cell proliferation, metastasis and apoptosis. SUMO can be used as a potential therapeutic target for cancer. In this review, we briefly outline the basic concepts of the SUMO system and summarize the involvement of SUMO proteins in cancer cells in order to better understand the role of SUMO in human disease.
... SUMOylation dramatically increases in response to HS. This mechanism, which is conserved in eukaryotes, plays an important role in protecting cells from stressderived injuries (Kurepa et al., 2003;Golebiowski et al., 2009). In Arabidopsis, chromatin-associated SUMOylation is induced by high temperature and modulates the transcriptional switch between plant development and HS responses (Han et al., 2021a). ...
Heat stress (HS) has serious negative effects on plant development and has become a major threat to agriculture. A rapid transcriptional regulatory cascade has evolved in plants in response to HS. Nuclear Factor‐Y (NF‐Y) complexes are critical for this mechanism, but how NF‐Y complexes are regulated remains unclear. In this study, we identified NF‐YC10 (NF‐Y subunit C10), a central regulator of the HS response in Arabidopsis thaliana, as a substrate of SUMOylation, an important post‐translational modification. Biochemical analysis showed that the SUMO ligase SIZ1 (SAP AND MIZ1 DOMAIN‐CONTAINING LIGASE1) interacts with NF‐YC10 and enhances its SUMOylation during HS. The SUMOylation of NF‐YC10 facilitates its interaction with and the nuclear translocation of NF‐YB3, in which the SUMO interaction motif (SIM) is essential for its efficient association with NF‐YC10. Further functional analysis indicated that the SUMOylation of NF‐YC10 and the SIM of NF‐YB3 are critical for HS‐responsive gene expression and plant thermotolerance. These findings uncover a role for the SIZ1‐mediated SUMOylation of NF‐YC10 in NF‐Y complex assembly under HS, providing new insights into the role of a post‐translational modification in regulating transcription during abiotic stress responses in plants.
... The present study was designed to identify SUMO2modified proteins and elucidate the cardioprotective mechanisms. SUMOylated proteins were successfully isolated by several strategies including SUMO antibody, an N-terminal tandem affinity protein tag, N-6xHis-SUMO transfection and GST-SIM fusion protein [42][43][44]. In the present study, we introduced the plasmid construct pcDNA3.1-N-Strep-SUMO2 ...
Myocardial infarction triggers oxidative DNA damage, apoptosis and adverse cardiac remodeling in the heart. Small ubiquitin-like modifier (SUMO) proteins mediate post-translational SUMOylation of the cardiac proteins in response to oxidative stress signals. Upregulation of isoform SUMO2 could attenuate myocardial injury via increasing protein SUMOylation. The present study aimed to discover the identity and cardioprotective activities of SUMOylated proteins. A plasmid vector for expressing N-Strep-SUMO2 protein was generated and introduced into H9c2 rat cardiomyocytes. The SUMOylated proteins were isolated with Strep-Tactin® agarose beads and identified by MALDI-TOF-MS technology. As a result, γ-actin was identified from a predominant protein band of ~42 kDa and verified by Western blotting. The roles of SUMO2 and γ-actin SUMOylation were subsequently determined in a mouse model of myocardial infarction induced by ligating left anterior descending coronary artery and H9c2 cells challenged by hypoxia-reoxygenation. In vitro lentiviral-mediated SUMO2 expression in H9c2 cells were used to explore the role of SUMOylation of γ-actin. SUMOylation of γ-actin by SUMO2 was proven to be a new cardioprotective mechanism from the following aspects: 1) SUMO2 overexpression reduced the number of TUNEL positive cells, the levels of 8-OHdG and p-γ-H2ax while promoted the nuclear deposition of γ-actin in mouse model and H9c2 cell model of myocardial infarction; 2) SUMO-2 silencing decreased the levels of nuclear γ-actin and SUMOylation while exacerbated DNA damage; 3) Mutated γ-actin (K68R/K284R) void of SUMOylation sites failed to protect cardiomyocytes against hypoxia-reoxygenation challenge. The present study suggested that SUMO2 upregulation promoted DNA damage repair and attenuated myocardial injury via increasing SUMOylation of γ-actin in the cell nucleus.
... Among the post-translation modifications, SUMOylation is a crucial mechanism, which senses the external challenges that a cell can go through and regulates the function of its target proteins accordingly [53]. There has been evidence showing global change in SUMOylation upon heat shock [54,55], starvation [56], osmotic stress [57], DNA damage and hypoxia. Depending on their cellular function, while some of the SUMO target proteins are less SUMOylated, others are increasingly SUMOylated on global stress. ...
Epigenetic mechanisms control chromatin accessibility and gene expression to ensure proper cell fate specification. Histone proteins are integral chromatin components, and their modification promotes gene expression regulation. Specific proteins recognize modified histones such as the chromodomain protein MRG-1. MRG-1 is the Caenorhabditis elegans ortholog of mammalian MRG15, which is involved in DNA repair. MRG-1 binds methylated histone H3 and is important for germline maturation and safeguarding. To elucidate interacting proteins that modulate MRG-1 activity, we performed in-depth protein–protein interaction analysis using immunoprecipitations coupled with mass spectrometry. We detected strong association with the Small ubiquitin-like modifier SUMO, and found that MRG-1 is post-translationally modified by SUMO. SUMOylation affects chromatin-binding dynamics of MRG-1, suggesting an epigenetic regulation pathway, which may be conserved.
... SUMOylation regulates numerous cellular processes, including transcription, chromatin organization, DNA repair, macromolecular assembly, and signal transduction (1). While SUMOylation has long been associated with stress responses, integrating a diverse array of cellular stress signals that trigger rapid increases in global protein SUMOylation (2)(3)(4)(5), how these cellular stresses promote SUMOylation remains a mystery. In addition, unlike ubiquitination that is tightly regulated by a large number of ubiquitin processing enzymes, including 2 E1 ubiquitin-activating enzymes, 30 to 50 E2 ubiquitin-conjugating enzymes and more than 600 E3 ubiquitin ligases (6), protein SUMOylation is controlled by a single pair of SUMO-activating enzyme (AOS1/UBA2) E1/ SUMO-conjugating enzyme (UBC9) E2 and a minimal set of validated E3 ligases (7). ...
Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, precisely how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation, remains elusive. Here, we report that cAMP, a major stress-response second messenger, acts through Epac1 as a regulator of cellular SUMOylation. The Epac1-associated proteome is highly enriched with components of the SUMOylation pathway. Activation of Epac1 by intracellular cAMP triggers phase separation and the formation of nuclear condensates containing Epac1 and general components of the SUMOylation machinery to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low-density lipoprotein-induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. These results provide a direct nexus connecting two major cellular stress responses to define a molecular mechanism in which cAMP regulates the dynamics of cellular condensates to modulate protein SUMOylation.
... Despite the fact RNA-related proteins are the most abundant group among SUMOylation substrates (56)(57)(58), including many snRNA-related factors, very little is known about the regulation of proteins involved in snRNA biogenesis by SUMO conjugation. Previous studies have shown that depletion of the SUMO protease USPL1 causes a reduction in nascent and mature snRNAs levels, diminishes snRNPs production, and alters pre-mRNA splicing (51). ...
RNA pol II transcribes snRNA genes in close proximity to Cajal bodies, subnuclear compartments that depend on the SUMO isopeptidase USPL1 for their assembly. We show here that over-expression of USPL1 alters snRNA 3-end cleavage, a process carried out by the Integrator complex. Beyond its role in snRNA biogenesis, this complex is responsible for regulating the expression of different non-coding and coding transcripts. We validated several subunits of the complex as SUMO conjugation substrates, and found that the SUMOylation of INTS11 subunit is regulated by USPL1. We defined Lys 381, Lys 462 and Lys 475 as bona fide SUMO attachment sites within INTS11 and observed that SUMOylation of this protein is required for efficient Integrator activity. Moreover, while an INTS11 SUMOylation deficient mutant is still capable of interacting with INTS4 and INTS9, its interaction with other subunits of the complex is affected. This mutant also shows a more cytoplasmatic localization than the wild type protein. These findings point to a regulatory role of SUMO conjugation on Integrator activity and suggest the involvement of INTS11 SUMOylation in the assembly of the complex. Furthermore, this work adds the Integrator-dependent RNA processing to the growing list of cellular processes regulated by SUMO conjugation.
