Nuclear Puncta Research Articles

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis.

Peptidyl-prolyl isomerase-like 1 (PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2 (AtCYP18-2), a PPIL1 homolog, plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions, AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP18-2 interacts with several spliceosome complex BACT components in nuclear puncta and is primarily associated with the small nuclear RNAs U5 and U6 in response to heat stress. The AtCYP18-2 loss-of-function allele cyp18-2 engineered by CRISPR/Cas9-mediated gene editing exhibited a hypersensitive phenotype to heat stress relative to the wild type. Moreover, global transcriptome profiling showed that the cyp18-2 mutation affects alternative splicing of heat stress-responsive genes under heat stress conditions, particularly intron retention (IR). The abundance of most intron-containing transcripts of a subset of genes essential for thermotolerance decreased in cyp18-2 compared to the wild type. Furthermore, the intron-containing transcripts of two heat stress-related genes, HEAT SHOCK PROTEIN 101 (HSP101) and HEAT SHOCK FACTOR A2 (HSFA2), produced functional proteins. HSP101-IR-GFP localization was responsive to heat stress, and HSFA2-III-IR interacted with HSF1 and HSP90.1 in plant cells. Our findings reveal that CYP18-2 functions as a splicing factor within the BACT spliceosome complex and is crucial for ensuring the production of adequate levels of alternatively spliced transcripts to enhance thermotolerance.

AML-158 Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation

Rearrangements of the nucleoporin 98 gene (NUP98) define a high-risk subset of childhood acute myeloid leukemia (AML). The resulting fusion oncoproteins (FOs) involve the N-terminal, intrinsically disordered region of NUP98, and the C-terminal portion of one of more than 30 identified fusion partners. Approximately one third of fusion partners have DNA-binding homeodomains, and the remaining partners have other domains involved in gene regulation. NUP98 FOs have long been known to localize in nuclear puncta. Here, we investigated whether these puncta form by liquid-liquid phase separation (LLPS) and how they might contribute to cell transformation. We first focused on the NUP98::HOXA9 (NHA9) FO, in which the HOXA9 fusion partner includes a DNA-binding homeodomain. We expressed GFP-tagged NHA9 in HEK293T cells and characterized the resulting FO-associated nuclear puncta. We also investigated lentiviral FO expression in mouse hematopoietic stem and progenitor cells (HSPCs), studying localization using confocal imaging, self-renewal using colony forming unit assays, and gene expression using RNA sequencing. We next mutated the phenylalanine glycine (FG) repeats of NUP98 to disrupt interactions with other FOs and interacting proteins or mutated the homeodomain of HOXA9 to disrupt DNA binding. Finally, we investigated the applicability of our findings to other NUP98 FOs with and without homeodomains. NHA9 localizes in nuclear puncta in HEK293T and HSPCs, and mutation of FG repeats or the HOXA9 homeodomain abrogates puncta formation. Furthermore, expression of NHA9 confers self-renewal, but cell transformation does not occur in FO mutants that fail to form puncta. Upregulation of NUP98 FO target genes, including the HOXA cluster, occurred with expression of NHA9 and transforming, puncta-positive mutants but not in mutant cells that did not form puncta or transform HSPCs. Expression of additional NUP98 FOs (including NUP98::KDM5A, NUP98::LNP1, and NUP98::PRRX1) led to both puncta formation and cell transformation. Finally, cells from a NUP98::KDM5A patient derived xenograft confirmed puncta formation with endogenous FO expression. Taken together, these results demonstrate that NUP98 FOs undergo LLPS, show that phase separation is critical for cell transformation and gene expression changes, and provide rationale to further explore the exploitation of NUP98 FO-associated puncta for therapeutical benefit.

Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression.

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.

Granule Cell Dispersion in Human Temporal Lobe Epilepsy: Proteomics Investigation of Neurodevelopmental Migratory Pathways.

Granule cell dispersion (GCD) is a common pathological feature observed in the hippocampus of patients with Mesial Temporal Lobe Epilepsy (MTLE). Pathomechanisms underlying GCD remain to be elucidated, but one hypothesis proposes aberrant reactivation of neurodevelopmental migratory pathways, possibly triggered by febrile seizures. This study aims to compare the proteomes of basal and dispersed granule cells in the hippocampus of eight MTLE patients with GCD to identify proteins that may mediate GCD in MTLE. Quantitative proteomics identified 1,882 proteins, of which 29% were found in basal granule cells only, 17% in dispersed only and 54% in both samples. Bioinformatics analyses revealed upregulated proteins in dispersed samples were involved in developmental cellular migratory processes, including cytoskeletal remodeling, axon guidance and signaling by Ras homologous (Rho) family of GTPases (P < 0.01). The expression of two Rho GTPases, RhoA and Rac1, was subsequently explored in immunohistochemical and in situ hybridization studies involving eighteen MTLE cases with or without GCD, and three normal post mortem cases. In cases with GCD, most dispersed granule cells in the outer-granular and molecular layers have an elongated soma and bipolar processes, with intense RhoA immunolabeling at opposite poles of the cell soma, while most granule cells in the basal granule cell layer were devoid of RhoA. A higher percentage of cells expressing RhoA was observed in cases with GCD than without GCD (P < 0.004). In GCD cases, the percentage of cells expressing RhoA was significantly higher in the inner molecular layer than the granule cell layer (P < 0.026), supporting proteomic findings. In situ hybridization studies using probes against RHOA and RAC1 mRNAs revealed fine peri- and nuclear puncta in granule cells of all cases. The density of cells expressing RHOA mRNAs was significantly higher in the inner molecular layer of cases with GCD than without GCD (P = 0.05). In summary, our study has found limited evidence for ongoing adult neurogenesis in the hippocampus of patients with MTLE, but evidence of differential dysmaturation between dispersed and basal granule cells has been demonstrated, and elevated expression of Rho GTPases in dispersed granule cells may contribute to the pathomechanisms underpinning GCD in MTLE.

Visualizing Structural Details of Disordered Domain Phase Separation Associated with ALS and Cancers

Phase separation of RNA-binding proteins via multivalent interactions between aromatic/polar-rich disordered domains contributes to the formation of functional cytoplasmic granules and nuclear puncta. These domains have also been identified as the nucleators of neuronal inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We use atomic resolution nuclear magnetic resonance spectroscopy approaches for visualizing low complexity domain structure and interactions along the pathway from monomer, to liquid-liquid phase separated state, to static aggregates and hydrogels. We show that the low complexity domain of RNA-binding protein Fused in Sarcoma (FUS LC) remains disordered even within liquid phase separated states and recruits unphosphorylated RNA-polymerase II C-terminal domain into the liquid phase separated state, adding a potential explanation for FUS LC transcriptional activation in cancer. Importantly, phase separation is reversible and is modulated by ionic strength and interaction with RNA, distinguishing these assemblies from static inclusions. In contrast, we show that liquid-liquid phase separation of TDP-43 is mediated in part by α-helical assembly and extension. Some ALS-associated mutations disrupt helix-helix interaction inhibiting liquid-liquid phase separation while leading to enhanced aggregation.

Impacts of chronic sleep deprivation on learning and memory, autophagy and neuronal apoptosis in mice

Objective To establish chronic sleep deprivation mouse model, evaluate the learning and memory ability of mice and observe autophagy and apoptosis levels in mouse brain. Methods C57BL/6 mice (n=20) were randomly separated into sleep deprivation group and control group. After 2-month sleep deprivation by using an adapted multiple platform method, the behavioral performance of mice was measured by IntelliCage system. The expression of microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) and Beclin-1 was detected by Western blotting. Confocol microscopy was used to observe autophagosome. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to detect neuronal apoptosis level in mouse brain. Results The results of behavioral test showed that the incorrect visit ratio was much higher in sleep deprivation group than that in control group. Moreover, the expression of LC3-Ⅱ(sleep deprivation group 1.681±0.186, control group 1.125±0.048, t=2.892, P=0.027 6) and Beclin-1(sleep deprivation group 1.144±0.048, control group 1.006±0.017, t=2.721, P=0.018 6) in mouse hippocampus and cortex was significantly elevated in sleep deprivation group than those in control group. Accordingly, the confocal microscopy observation also revealed an increased nuclear LC3-positive puncta in hippocampus and cortex of sleep deprived mice (hippocampus in sleep deprivation group 1.665±0.153, in control group 0.819±0.072, t=5.024, P=0.002 4; cortex in sleep deprivation group 1.925±0.175, in control group 1.195±0.111, t=3.521, P=0.012 5). In addition, TUNEL staining showed a much higher percentage of TUNEL-positive nuclei in these brain regions (hippocampus in sleep deprivation group 47.24±4.15, in control group 19.26±3.72, t=5.025, P=0.007 4; cortex in sleep deprivation group 42.25±1.25, in control group 27.50±3.23, t=4.262, P=0.005 3). Conclusions Chronic sleep deprivation can impair the learning and memory, increase the expression of LC3-Ⅱ and Beclin-1, elevate the formation of autophagosome, and promote apoptosis in mouse brain. These findings suggest that autophagy and apoptosis might be involved in the cognitive impairment induced by chronic sleep deprivation. Key words: Sleep deprivation; Neurons; Autophagy; Apoptosis; Memory disorders; Disease models, animal

The Crz1/Sp1 Transcription Factor of Cryptococcus neoformans Is Activated by Calcineurin and Regulates Cell Wall Integrity

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δcna1, and a CRZ1/SP1 mutant (Δcrz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and Δcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30–37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.

Uncovering a Mitochondrial Unfolded Protein Response

Proteins are synthesized and folded and assembled in the cytosol, endoplasmic reticulum (ER), and mitochondria. For the cytosol and ER, pathways associated with excess unfolded or misfolded proteins have been characterized and are called the heat shock response and the ER unfolded protein response (UPR er ). Haynes et al . applied a genome-wide RNAi screen to identify four genes involved in the mitochondrial UPR (UPR mt ) in Caenorhabditis elegans . Three of the genes were characterized in detail: dve-1 , which encodes a homolog of the Drosophila homeobox transcription factor defective proventriculus and mammalian SatB2; ubl - 5 , which encodes a ubiquitin-like protein; and clpp-1 , which encodes a protein homologous to the bacterial protease ClpP. (The fourth encoded guanosine triphosphatase homologous to mammalian Rheb.) Reduction in the expression of any of these genes compromised the expression of the chaperone Hsp60 in response to mitochondrial unfolded protein stress without interfering with UPR er and caused perturbed mitochondrial morphology, consistent with persistent UPR mt . DVE-1 showed a nuclear localization and redistributed to nuclear puncta in response to conditions of mitochondrial unfolded protein stress. In worms deficient in, but not completely lacking, DVE-1 (the latter was lethal), mitochondrial mass was decreased (as detected by MitoTracker staining and ligand blot assay). In an affinity purification assay, a tagged form of DVE-1 and a tagged form of UBL-5 were found to form a complex when the worms were subjected to conditions causing mitochondrial unfolded protein stress. A similar interaction was observed for the mammalian homologs. RNAi experiments suggested that CLPP-1 acted upstream of UBL-5 (the expression of which is stimulated in response to conditions of mitochondrial unfolded protein stress) and of DVE-1 in the UPR mt . CLPP-1 was localized to the matrix of the mitochondria, where it appeared to be part of a large multisubunit complex. When CLPP-1 activity was decreased by RNAi, increased expression of the ubl-5 and hsp60 genes in response to conditions that cause mitochondrial unfolded protein stress was compromised, and the redistribution of DVE-1 to nuclear puncta was also blocked. Protease inhibitors also prevented the stimulation of ubl-5 expression in response to mitochondrial unfolded protein stress, suggesting that the protease activity of CLPP-1 is important for its function in the UPR mt . C. M. Haynes, K. Petrova, C. Benedetti, Y. Yang, D. Ron, ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans . Dev. Cell 13 , 467-480 (2007). [PubMed]