Galectin-3-U1 snRNP Complexes Initiate Splicing Activity in U1-Depleted Nuclear Extracts.

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.

An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites.

Proliferation of Plasmodium falciparum in red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms, P. falciparum replicates by multiple rounds of nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of critical nuclear division factors remain poorly understood. Centriolar plaques, the centrosomes of P. falciparum, serve as microtubule organizing centers and have an acentriolar, amorphous structure. The small size of parasite nuclei has precluded detailed analysis of intranuclear microtubule organization by classical fluorescence microscopy. We apply recently developed super-resolution and time-lapse imaging protocols to describe microtubule reconfiguration during schizogony. Analysis of centrin, nuclear pore, and microtubule positioning reveals two distinct compartments of the centriolar plaque. Whereas centrin is extranuclear, we confirm by correlative light and electron tomography that microtubules are nucleated in a previously unknown and extended intranuclear compartment, which is devoid of chromatin but protein-dense. This study generates a working model for an unconventional centrosome and enables a better understanding about the diversity of eukaryotic cell division.

A nervous system-specific subnuclear organelle in Caenorhabditis elegans.

We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4-10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

De novo TRIM8 variants impair its protein localization to nuclear bodies and cause developmental delay, epilepsy, and focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

Nuclear Phosphoinositides: Their Regulation and Roles in Nuclear Functions.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

Nab3's localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain.

Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3's proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro. Localization to the granule is reversible and sensitive to the chemical probe 1,6 hexanediol suggesting condensation is driven by phase separation. Nab3's RNA recognition motif is also required for localization as seen for other PrLD-containing RNA-binding proteins that phase separate. Although the PrLD is necessary, it is not sufficient to localize to the granule. A heterologous PrLD that functionally replaces Nab3's essential PrLD, directed localization to the nuclear granule, however a chimeric Nab3 molecule with a heterologous PrLD that cannot restore termination function or viability, does not form granules. The Nab3 nuclear granule shows properties similar to well characterized cytoplasmic compartments formed by phase separation, suggesting that, as seen for other elements of the transcription machinery, termination factor condensation is functionally important.

Intronless mRNAs transit through nuclear speckles to gain export competence.

Nuclear speckles (NSs) serve as splicing factor storage sites. In this study, we unexpectedly found that many endogenous intronless mRNAs, which do not undergo splicing, associate with NSs. These associations do not require transcription, polyadenylation, or the polyA tail. Rather, exonic splicing enhancers present in intronless mRNAs and their binding partners, SR proteins, promote intronless mRNA localization to NSs. Significantly, speckle targeting of mRNAs promotes the recruitment of the TREX export complex and their TREX-dependent nuclear export. Furthermore, TREX, which accumulates in NSs, is required for releasing intronless mRNAs from NSs, whereas NXF1, which is mainly detected at nuclear pores, is not. Upon NXF1 depletion, the TREX protein UAP56 loses speckle concentration but coaccumulates with intronless mRNAs and polyA RNAs in the nucleoplasm, and these RNAs are trapped in NSs upon UAP56 codepletion. We propose that the export-competent messenger RNP assembly mainly occurs in NSs for intronless mRNAs and that entering NSs serves as a quality control step in mRNA export.

Intranuclear delivery of synthetic nuclear factor-kappa B p65 reduces inflammasomes after surgery.

Patients undergoing surgery can suffer from various complications, including post-operative bleeding, local or systematic infection, and neurologic disorders. Major surgery can initiate innate immune responses and trigger overproduction of inflammatory mediators, which can contribute to organ dysfunction. Inflammasomes are innate immune complexes, which are connected to the pathogenesis of various diseases, including atherosclerosis, hemorrhagic brain injury, and Alzheimer's disease. In the present study, we hypothesized that nucleotide-binding oligomerization domain-containing-like receptor protein (NLRP) inflammasomes may have a role in the pathological effects of surgery. Therefore, we designed a protein inhibitor of nuclear factor kappa B (NF-κB) p65 transcripts, called nt-p65-TMD (nuclear transducible (nt) transcription modulated domain (TMD) of RelA (p65)), that can penetrate the nucleus, and evaluated its therapeutic efficacy for dampening surgery-induced inflammasome activation. It was found that the nt-p65-TMD significantly reduced the NLRP1 inflammasome complex components (NLRP1, ASC, and Caspase-1) and interleukin (IL)-1ß and IL-18 productions in the spleen after surgery. In the spleen, specific cell population and selective mediators were altered after surgery with/without nt-p65-TMD treatment. Also, we found that treatment of nt-p65-TMD decreased cell death in the spleen after surgery. Therefore, nt-p65-TMD is a potential novel strategy for reducing surgery-induced NLRP1 inflammasome and complications.

Quality Control of Membraneless Organelles.

The formation of membraneless organelles (MLOs) by phase separation has emerged as a new way of organizing the cytoplasm and nucleoplasm of cells. Examples of MLOs forming via phase separation are nucleoli in the nucleus and stress granules in the cytoplasm. The main components of these MLOs are macromolecules such as RNAs and proteins. In order to assemble by phase separation, these proteins and RNAs have to undergo many cooperative interactions. These cooperative interactions are supported by specific molecular features within phase-separating proteins, such as multivalency and the presence of disordered domains that promote weak and transient interactions. However, these features also predispose phase-separating proteins to aberrant behavior. Indeed, evidence is emerging for a strong link between phase-separating proteins, MLOs, and age-related diseases. In this review, we discuss recent progress in understanding the formation, properties, and functions of MLOs. We pay special attention to the emerging link between MLOs and age-related diseases, and we explain how changes in the composition and physical properties of MLOs promote their conversion into an aberrant state. Furthermore, we discuss the key role of the protein quality control machinery in regulating the properties and functions of MLOs and thus in preventing age-related diseases.

C-terminal short arginine/serine repeat sequence-dependent regulation of Y14 (RBM8A) localization.

Y14 (RBM8A) is an RNA recognition motif-containing protein that forms heterodimers with MAGOH and serves as a core factor of the RNA surveillance machinery for the exon junction complex (EJC). The role of the Y14 C-terminal serine/arginine (RS) repeat-containing region, which has been reported to undergo modifications such as phosphorylation and methylation, has not been sufficiently investigated. Thus, we aimed to explore the functional significance of the Y14 C-terminal region. Deletion or dephosphorylation mimic mutants of the C-terminal region showed a shift in localization from the nucleoplasmic region; in addition, the C-terminal RS repeat-containing sequence itself exhibited the potential for nucleolar localization. Additionally, the regulation of Y14 localization by the C-terminal region was further found to be exquisitely controlled by MAGOH binding. Cumulatively, our findings, which demonstrated that Y14 localization is regulated not only by the previously reported N-terminal localization signal but also by the C-terminal RS repeat-containing region through phosphorylation and MAGOH binding to Y14, provide new insights for the mechanism of localization of short RS repeat-containing proteins.

The functional roles of PML nuclear bodies in genome maintenance.

In the nucleus, there are several membraneless structures called nuclear bodies. Among them, promyelocytic leukemia nuclear bodies (PML-NBs) are involved in multiple genome maintenance pathways including the DNA damage response, DNA repair, telomere homeostasis, and p53-associated apoptosis. In response to DNA damage, PML-NBs are coalesced and divided by a fission mechanism, thus increasing their number. PML-NBs also play a role in repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). Clinically, the dominant negative PML-RARα fusion protein expressed in acute promyelocytic leukemia (APL) inhibits the transactivation of downstream factors and disrupts PML function, revealing the tumor suppressor role of PML-NBs. All-trans retinoic acid and arsenic trioxide treatment has been implemented for promyelocytic leukemia to target the PML-RARα fusion protein. PML-NBs are associated with various factors implicated in genome maintenance, and are found at the sites of DNA damage. Their interaction with proteins such as p53 indicates that PML-NBs may play a significant role in apoptosis and cancer. Decades of research have revealed the importance of PML-NBs in diverse cellular pathways, yet the underlying molecular mechanisms and exact functions of PML-NBs remain elusive. In this review, PML protein modifications and the functional relevance of PML-NB and its associated factors in genome maintenance will be discussed.

Chromatin topology is coupled to Polycomb group protein subnuclear organization.

The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.

Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA.

An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one.

Nucleus accumbens associated 1 is recruited within the promyelocytic leukemia nuclear body through SUMO modification.

Nucleus accumbens associated 1 (NACC1) is a cancer-associated BTB/POZ (pox virus and zinc finger/bric-a-brac tramtrack broad complex) gene, and is involved in several cellular functions in neurons, cancer and stem cells. Some of the BTB/POZ proteins associated with cancer biology are SUMOylated, which appears to play an important role in transcription regulation. We show that NACC1 is SUMOylated on a phylogenetically conserved lysine (K167) out of three consensus SUMOylation motif sites. Amino acid substitution in the SIM sequence (SIM/M) within the BTB/POZ domain partially reduced K167 SUMOylation activity of NACC1. Overexpression of GFP-NACC1 fusion protein leads to formation of discrete nuclear foci similar to promyelocytic leukemia nuclear bodies (PML-NB), which colocalized with SUMO paralogues (SUMO1/2/3). Both NACC1 nuclear body formation and colocalization with SUMO paralogues were completely suppressed in the GFP-NACC1-SIM/M mutant, whereas they were partially maintained in the NACC1 K167R mutant. Confocal immunofluorescence analysis showed that endogenous and exogenous NACC1 proteins colocalized with endogenous PML protein. A pull-down assay revealed that the consensus motifs of the SUMO acceptor site at K167 and the SIM within the BTB/POZ domain were both necessary for efficient binding to PML protein. Our study demonstrates that NACC1 can be modified by SUMO paralogues, and cooperates with PML protein.

Mechanistic target of rapamycin complex 1 is critical for invariant natural killer T-cell development and effector function.

The mechanisms that control invariant natural killer T (iNKT)-cell development and function are still poorly understood. The mechanistic or mammalian target of rapamycin (mTOR) integrates various environmental signals/cues to regulate cell growth, proliferation, metabolism, and survival. We report here that ablation of mTOR complex 1 (mTORC1) signaling by conditionally deleting Raptor causes severe defects in iNKT-cell development at early stages, leading to drastic reductions in iNKT-cell numbers in the thymus and periphery. In addition, loss of Raptor impairs iNKT-cell proliferation and production of cytokines upon α-galactosylceramide stimulation in vitro and in vivo, and inhibits liver inflammation in an iNKT cell-mediated hepatitis model. Furthermore, Raptor deficiency and rapamycin treatment lead to aberrant intracellular localization and functional impairment of promyelocytic leukemia zinc-finger, a transcription factor critical for iNKT-cell development and effector programs. Our findings define an essential role of mTORC1 to direct iNKT-cell lineage development and effector function.

[Cajal bodies and histone locus bodies: molecular structure and function].

The review provides modern classification of evolutionarily conserved coilin-containing nuclear bodies of somatic and germ cells that is based on the characteristic features of their molecular composition and the nature of their functions. The main differences between Cajal bodies and histone locus bodies, which are involved in the biogenesis of small nuclear spliceosomal and nucleolar RNAs and in the 3'-end processing of histone precursor messenger RNA, respectively, are considered. It is shown that a significant contribution to the investigation of the diversity of coilin-containing bodies was made by the studies on the architecture of the RNA processing machinery in oocyte nuclei in a number of model organisms. The characteristics features of the molecular composition of coilin-containing bodies in the nuclei of growing oocytes (the so-called germinal vesicles) of vertebrates, including amphibians and birds, are described.

Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat.

Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNA-binding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain.

Postnatal development of Homer1a in the rat hippocampus.

Homer1a (H1a) is an immediate early gene involved in multiple forms of synaptic plasticity. It exhibits a postnatal increase in the rat forebrain (Brakeman et al. (1997) Nature 386:284-288) and reduces the density and size of dendritic spines in hippocampal neurons (Sala et al. (2003) J Neurosci 23:6327-6337). We evaluated hippocampal H1a expression at different postnatal ages (P3, P5, P7, P9, P15, P19, P23, P35, and adult) using Fluorescence In Situ Hybridization (FISH) and qRT-PCR. Maximal electroconvulsive shock (MECS) was used to induce maximal expression relative to home cage (HC) controls. Large scale images and confocal z-stacks from dorsal subiculum (DS), CA1, CA3, and dentate gyrus (DG) were analyzed by both manual and automated methods. In DS, CA1, and CA3 a significant proportion of cells (40%) expressed small but detectable levels of H1a from P3; however, MECS did not up-regulate H1a during the first postnatal week. MECS induced H1a positive cells during the second postnatal week and induction reached adult levels at P9. H1a-Intra Nuclear Foci (INF) size and intensity varied with age, increasing at P19-23 in CA1 and CA3 and from P9 to P23 in DS. In DG, H1a expression exhibited a lamination pattern and an H1a-INF size and intensity gradient across the granule cell layer, consistent with the outside-in maturation of DG granule cells. The developmental progression of H1a corresponds to the synaptic refinement period supporting the conclusion that H1a could play an important role in this process.

Myeloma overexpressed 2 (Myeov2) regulates L11 subnuclear localization through Nedd8 modification.

Nucleolus is a dynamic structure that controls biogenesis of ribosomal RNA and senses cellular stresses. Nucleolus contains a number of proteins including ribosomal proteins that conduct cellular stresses to downstream signaling such as p53 pathway. Recently, it has been reported that modification by a ubiquitin-like molecule, Nedd8, regulates subnuclear localization of ribosomal protein L11. Most of L11 is normally localized and neddylated in nucleolus. However, cellular stress triggers deneddylation and redistribution of L11, and subsequent activation of p53. Although Nedd8 modification is thought to be important for L11 localization, the mechanism of how neddylation of L11 is regulated remains largely unknown. Here, we show that Myeloma overexpressed 2 (Myeov2) controls L11 localization through down-regulation of Nedd8 modification. Expression of Myeov2 reduced neddylation of proteins including L11. We also found that Myeov2 associates with L11 and withholds L11 in nucleoplasm. Although Myeov2 interacted with a Nedd8 deconjugation enzyme COP9 signalosome, L11 deneddylation was mediated by another deneddylase Nedp1, independently of Myeov2. Finally, p53 transcriptional activity is upregulated by Myeov2 expression. These data demonstrate that Myeov2 hampers L11 neddylation through their interactions and confines L11 to nucleoplasm to modulate nucleolar integrity. Our findings provide a novel link between oncogenic stress and p53 pathway and may shed light on the protective mechanism against cancer.

Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation.

BACKGROUND: The Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4⁺ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions. RESULTS: In this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4⁺ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation. CONCLUSIONS: These data reveal that the formation of Tax nuclear bodies, previously associated to transcriptional activities in Tax-transfected cells, is dispensable for NF-κB promoter activation, notably in CD4⁺ T cells. They also provide the first evidence that Tax SUMOylation is not a key determinant for Tax-induced NF-κB activation.