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IKKα is known as an essential protein for skin homeostasis. However, the lack of suitable models to investigate its functions in the skin has led to IKKα being mistakenly considered as a suppressor of non-melanoma skin cancer (NMSC) development. In this study, using our previously generated transgenic mouse models expressing exogenous IKKα in the cytoplasm (C-IKKα mice) or in the nucleus (N-IKKα mice) of basal keratinocytes, we demonstrate that at each subcellular localization, IKKα differently regulates signaling pathways important for maintaining the balance between keratinocyte proliferation and differentiation, and for the cutaneous inflammatory response. In addition, each type of IKKα-transgenic mice shows different predisposition to the development of spontaneous NMSC. Specifically, N-IKKα mice display an atrophic epidermis with exacerbated terminal differentiation, signs of premature skin aging, premalignant lesions, and develop squamous cell carcinomas (SCCs). Conversely, C-IKKα mice, whose keratinocytes are nearly devoid of endogenous nuclear IKKα, do not develop skin SCCs, although they exhibit hyperplastic skin with deficiencies in terminal epidermal differentiation, chronic cutaneous inflammation, and constitutive activation of STAT-3 and NF-κB signaling pathways. Altogether, our data demonstrate that alterations in the localization of IKKα in the nucleus or cytoplasm of keratinocytes cause opposite skin changes and differentially predispose to the growth of skin SCCs., Competing Interests: Competing interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© 2024. The Author(s).)

Dopamine (DA) plays important roles in various behaviors, including learning and motivation. Recently, THOC5 was identified as an important regulator in the development of dopaminergic neurons. However, how THOC5 is regulated has not been explored. In this study, we found an interaction between THOC5 and necdin, which is encoded by a gene located in the chromosome deletion region of Prader-Willi syndrome (PWS), by using a yeast two-hybrid assay. Necdin affects the mRNA export function of THOC5 by regulating its nucleocytoplasmic localization. As a result, the expression of a few DA neuronal development-related genes, such as Mef2c, Lef1 and Prkcg, is altered in necdin-deficient mice. We also found neurodegeneration of dopaminergic neurons and an increase of glial cells in necdin-deficient mice, which may underlie the dyspraxia behaviors in these mice. Our results thus identified necdin as a novel regulator for THOC5, which may underlie, at least partly, the abnormal DA neuron development in necdin-deficient mice., Competing Interests: Declarations. Ethics approval: All procedures regarding the care and use of animals were approved by the ethics committee of Center for Medical Genetics, School of Life Sciences, Central South University of China. All methods were performed in accordance with approved guidelines. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

N6-methyladenosine (m 6 A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology-domain family (YTHDF) proteins. Here, we show that in Arabidopsis thaliana, the interaction between m 6 A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins and their mRNA target sets overlap, with ALBA4 binding sites being juxtaposed to m 6 A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m 6 A binding to ECT2. Accordingly, both the biological functions of ECT2, and its binding to m 6 A targets in vivo, require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m 6 A-reader in Arabidopsis, and define a molecular foundation for the concept of facilitated m 6 A reading, which increases the potential for combinatorial control of biological m 6 A effects., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The interplay between translation and mRNA decay is widespread in human cells 1-3 . In quality-control pathways, exonucleolytic degradation of mRNA associated with translating ribosomes is mediated largely by the cytoplasmic exosome 4-9 , which includes the exoribonuclease complex EXO10 and the helicase complex SKI238 (refs. 10-16 ). The helicase can extract mRNA from the ribosome and is expected to transfer it to the exoribonuclease core through a bridging factor, HBS1L3 (also known as SKI7), but the mechanisms of this molecular handover remain unclear 7,17,18 . Here we reveal how human EXO10 is recruited by HBS1L3 (SKI7) to an active ribosome-bound SKI238 complex. We show that rather than a sequential handover, a direct physical coupling mechanism takes place, which culminates in the formation of a cytoplasmic exosome-ribosome supercomplex. Capturing the structure during active decay reveals a continuous path in which an RNA substrate threads from the 80S ribosome through the SKI2 helicase into the exoribonuclease active site of the cytoplasmic exosome complex. The SKI3 subunit of the complex directly binds to HBS1L3 (SKI7) and also engages a surface of the 40S subunit, establishing a recognition platform in collided disomes. Exosome and ribosome thus work together as a single structural and functional unit in co-translational mRNA decay, coordinating their activities in a transient supercomplex., (© 2024. The Author(s).)

The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response is mediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further show that the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer., (© 2024. The Author(s).)

Subcellular protein localization regulates protein function and can be corrupted in cancers 1 and neurodegenerative diseases 2,3 . The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1 Q318X , TDP43 ΔNLS and FUS R495X . TRAM-mediated relocalization of FUS R495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration 4 . The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67-Mtr2 in the yeast Saccharomyces cerevisiae (TAP-p15 in humans) 1,2 . Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm 3 . Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export., (© 2024. The Author(s).)

Biophysical cues from the cell microenvironment are detected by mechanosensitive components at the cell surface. Such machineries convert physical information into biochemical signaling cascades within cells, subsequently leading to various cellular responses in a stimulus-dependent manner. At the surface of extracellular environment and cell cytoplasm exist several ion channel families that are activated by mechanical signals to direct intracellular events. One of such channel is formed by transient receptor potential cation channel subfamily V member, TRPV4 that is known to act as a mechanosensor in wide variaty of tissues and control ion-influx in a spatio-temporal way. Here we report that TRPV4 is prominently expressed in the stem/progenitor cell populations of the mammary epithelium and seems important for the lineage-specific differentiation, consequently affecting mechanical features of the mature mammary epithelium. This was evident by the lack of several markers for mature myoepithelial and luminal epithelial cells in TRPV4-depleted cell lines. Interestingly, TRPV4 expression is controlled in a tension-dependent manner and it also impacts differentation process dependently on the stiffness of the microenvironment. Furthermore, such cells in a 3D compartment were disabled to maintain normal mammosphere structures and displayed abnormal lumen formation, size of the structures and disrupted cellular junctions. Mechanosensitive TRPV4 channel therefore act as critical player in the homeostasis of normal mammary epithelium through sensing the physical environment and guiding accordingly differentiation and structural organization of the bilayered mammary epithelium., (© 2024. The Author(s).)

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2) 1,2 , which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission 1-3 . Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression 4-6 . Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease 7,8 , respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP., (© 2023. The Author(s).)

Microsatellite repeat expansions within genes contribute to a number of neurological diseases 1,2 . The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology 3-9 . Here we reveal that the adenosine in CAG repeat RNA can be methylated to N 1 -methyladenosine (m 1 A) by TRMT61A, and that m 1 A can be demethylated by ALKBH3. We also observed that the m 1 A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m 1 A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m 1 A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m 1 A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion., (© 2023. The Author(s).)

Although haemoglobin is a known carrier of oxygen in erythrocytes that functions to transport oxygen over a long range, its physiological roles outside erythrocytes are largely elusive 1,2 . Here we found that chondrocytes produced massive amounts of haemoglobin to form eosin-positive bodies in their cytoplasm. The haemoglobin body (Hedy) is a membraneless condensate characterized by phase separation. Production of haemoglobin in chondrocytes is controlled by hypoxia and is dependent on KLF1 rather than the HIF1/2α pathway. Deletion of haemoglobin in chondrocytes leads to Hedy loss along with severe hypoxia, enhanced glycolysis and extensive cell death in the centre of cartilaginous tissue, which is attributed to the loss of the Hedy-controlled oxygen supply under hypoxic conditions. These results demonstrate an extra-erythrocyte role of haemoglobin in chondrocytes, and uncover a heretofore unrecognized mechanism in which chondrocytes survive a hypoxic environment through Hedy., (© 2023. The Author(s).)

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors. PGLs can further be divided into sympathetic (sPGLs) and head-and-neck (HN-PGLs). There are virtually no treatment options, and no cure, for metastatic PCCs and PGLs (PPGLs). Here, we composed a tissue microarray (TMA) consisting of 149 PPGLs, reflecting clinical features, presenting as a useful resource. Mutations in the pseudohypoxic marker HIF-2α correlate to an aggressive tumor phenotype. We show that HIF-2α localized to the cytoplasm in PPGLs. This subcompartmentalized protein expression differed between tumor subtypes, and strongly correlated to proliferation. Half of all sPGLs were metastatic at time of diagnosis. Cytoplasmic HIF-2α was strongly expressed in metastatic sPGLs and predicted poor outcome in this subgroup. We propose that higher cytoplasmic HIF-2α expression could serve as a useful clinical marker to differentiate paragangliomas from pheochromocytomas, and may help predict outcome in sPGL patients., (© 2023. The Author(s).)

IRF5, a nucleoplasm shuttling protein, is a pivotal transcription factor regulating immune system activity. It's well known that immunosuppression is involved in the development of gastric cancer. However, no data exist for the expression and function of IRF5 in gastric cancer. This study demonstrated that IRF5 was cytoplasm-enriched in gastric cancer cells. IRF5 promoted gastric cancer cell migration, which involved the inhibition of Wnt5a and E-cadherin proteins expression. IRF5 (LA) localized in nucleus had no significant effect on Wnt5a and E-cadherin expressions, while mutation of IRF5 (ΔNLS), which prevents IRF5 nuclear translocation, had more impact on these inhibitory effects. In addition, degradation rates of both Wnt5a and E-cadherin were enhanced by resiquimod, an IRF5 agonist. Further in vivo experiments indicated that IRF5 knockout of gastric cancer cells repressed their pulmonary metastasis in nude mice. Finally, the expression and clinical significance of IRF5 were analyzed using gastric cancer tissue microarrays, which suggested that the expression of IRF5 varied procedurally in different progressive stages of gastric cancer. Our data revealed that IRF5 cytoplasmic localization were associated with Wnt5a and E-cadherin degradation and gastric cancer cell metastasis. Inhibiting IRF5 expression and/or its cytoplasmic localization may provide a novel target for gastric cancer therapy., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae. While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae., (© 2023. The Author(s).)

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid-liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models., (© 2023. The Author(s).)

In human cells two dUTPase isoforms have been described: one nuclear (DUT-N) and one mitochondrial (DUT-M), with cognate localization signals. In contrast, here we identified two additional isoforms; DUT-3 without any localization signal and DUT-4 with the same nuclear localization signal as DUT-N. Based on an RT-qPCR method for simultaneous isoform-specific quantification we analysed the relative expression patterns in 20 human cell lines of highly different origins. We found that the DUT-N isoform is expressed by far at the highest level, followed by the DUT-M and the DUT-3 isoform. A strong correlation between expression levels of DUT-M and DUT-3 suggests that these two isoforms may share the same promoter. We analysed the effect of serum starvation on the expression of dUTPase isoforms compared to non-treated cells and found that the mRNA levels of DUT-N decreased in A-549 and MDA-MB-231 cells, but not in HeLa cells. Surprisingly, upon serum starvation DUT-M and DUT-3 showed a significant increase in the expression, while the expression level of the DUT-4 isoform did not show any changes. Taken together our results indicate that the cellular dUTPase supply may also be provided in the cytoplasm and starvation stress induced expression changes are cell line dependent., (© 2023. The Author(s).)

Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges. Here, we report cost and time-effective soluble production of SARS-CoV-2 receptor binding domain (RBD) variants as well as an array of neutralizing antibody fragments (Fabs) based on Casirivimab and Imdevimab using the CyDisCo system in the cytoplasm of E. coli. We also report variants of the two Fabs with higher binding affinity against SARS-CoV-2 RBD and suggest this cytoplasmic production of disulfide containing antigens and antibodies can be broadly applied towards addressing future global public health threats., (© 2023. The Author(s).)

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability 1 , which has been linked to the action of endonucleases such as XPG 2-4 . However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7 ), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref.  8 ), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref.  9 ) and in BRCA1-mutated cancer cells 10 . These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The fluorescent proteins superfolder mTurquoise2 ox (sfTq2 ox ) and mNeonGreen function excellently in mammalian cells, but are not well expressed in E. coli when forming the N-terminus of constructs. Expression was increased by decreasing structures at the start of their coding sequences in the mRNA. Unfortunately, the expression of mNeonGreen started from methionine at position ten as optimisation introduced an alternative RBS in the GFP N-terminus of mNeonGreen. The original start-codon was not deleted, which caused the expression of isomers starting at the original start-codon and at the start-codon at the beginning of the GFP N-terminus. By omitting the GFP N-terminus of mNeonGreen and optimising the structure of its mRNA, the expression of a mixture of isomers was avoided, and up to ~ 50-fold higher expression rates were achieved for mNeonGreen. Both fluorescent proteins can now be expressed at readily detectable levels in E. coli and can be used for various purposes. One explored application is the detection of in-cell protein interactions by FRET. mNeonGreen and sfTq2 ox form a FRET pair with a larger dynamic range than commonly used donor-acceptor pairs, allowing for an excellent signal-to-noise ratio, which supports the detection of conformational changes that affect the distance between the interacting proteins., (© 2022. The Author(s).)

Bucky ball was identified as germ plasm organizer in zebrafish and has proven crucial for Balbiani body condensation. A synteny comparison identified an uncharacterized gene locus in the chicken genome as predicted avian counterpart. Here, we present experimental evidence that this gene locus indeed encodes a 'Bucky ball' equivalent in matured oocytes and early embryos of chicken. Heterologous expression of Bucky ball fusion proteins both from zebrafish and chicken with a fluorescent reporter revealed unique patterns indicative for liquid-liquid phase separation of intrinsically disordered proteins. Immuno-labeling detected Bucky ball from oocytes to blastoderms with diffuse distribution in matured oocytes, aggregation in first cleavage furrows, and co-localization to the chicken vasa homolog (CVH). Later, Bucky ball translocated to the cytoplasm of first established cells, and showed nuclear translocation during the major zygotic activation together with CVH. Remarkably, during the phase of area pellucida formation, Bucky ball translocated back into the cytoplasm at stage EGK VI, whereas CVH remained within the nuclei. The condensation of Bucky ball and co-localization with CVH in cleavage furrows and nuclei of the centrally located cells strongly suggests chicken Bucky ball as a germ plasm organizer in birds, and indicate a special importance of the major zygotic activation for germline specification., (© 2022. The Author(s).)

Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease 1-4 . Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridges 5-8 . These structures are associated with fragile nuclear envelopes that spontaneously rupture 9,10 , leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by adenine deaminases acting on RNA (ADAR enzymes) to generate deoxyinosine. Deoxyinosine is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, N-methyl-purine DNA glycosylase 11,12 (MPG). These abasic sites are cleaved by the BER endonuclease, apurinic/apyrimidinic endonuclease 12 (APE1), creating single-stranded DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands 13,14 . This model predicts that MPG should be able to remove the deoxyinosine base from the DNA strand of RNA-DNA hybrids, which we demonstrate using purified proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with pre-existing defects such as the DNA base abnormality described here., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Fused in Sarcoma (FUS) is a nuclear RNA/DNA binding protein that mislocalizes to the cytoplasm in the neurodegenerative diseases ALS and FTD. Despite the existence of FUS pathogenic mutations that result in nuclear import defects, a subset of ALS/FTD patients display cytoplasmic accumulation of wild-type FUS, although the underlying mechanism is unclear. Here we confirm that transcriptional inhibition, specifically of RNA polymerase II (RNAP II), induces FUS cytoplasmic translocation, but we show that several other stresses do not. We found unexpectedly that the epitope specificity of different FUS antibodies significantly affects the apparent FUS nucleocytoplasmic ratio as determined by immunofluorescence, explaining inconsistent observations in previous studies. Significantly, depletion of the nuclear mRNA export factor NXF1 or RNA exosome cofactor MTR4 promotes FUS nuclear retention, even when transcription is repressed, while mislocalization was independent of the nuclear protein export factor CRM1 and import factor TNPO1. Finally, we report that levels of nascent RNAP II transcripts, including those known to bind FUS, are reduced in sporadic ALS iPS cells, linking possible aberrant transcriptional control and FUS cytoplasmic mislocalization. Our findings thus reveal that factors that influence accumulation of nuclear RNAP II transcripts modulate FUS nucleocytoplasmic homeostasis, and provide evidence that reduced RNAP II transcription can contribute to FUS mislocalization to the cytoplasm in ALS., (© 2022. The Author(s).)

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development., (© 2022. The Author(s).)

Nuclear pore complex (NPC) shuttles cargo across the nuclear envelope. Here we present single-particle cryo-EM structure of the nuclear ring (NR) subunit from Xenopus laevis NPC at an average resolution of 5.6 Å. The NR subunit comprises two 10-membered Y complexes, each with the nucleoporin ELYS closely associating with Nup160 and Nup37 of the long arm. Unlike the cytoplasmic ring (CR) or inner ring (IR), the NR subunit contains only one molecule each of Nup205 and Nup93. Nup205 binds both arms of the Y complexes and interacts with the stem of inner Y complex from the neighboring subunit. Nup93 connects the stems of inner and outer Y complexes within the same NR subunit, and places its N-terminal extended helix into the axial groove of Nup205 from the neighboring subunit. Together with other structural information, we have generated a composite atomic model of the central ring scaffold that includes the NR, IR, and CR. The IR is connected to the two outer rings mainly through Nup155. This model facilitates functional understanding of vertebrate NPC., (© 2022. The Author(s).)

A repeat expansion in C9orf72 is the major cause of both frontotemporal dementia and amyotrophic lateral sclerosis, accounting for approximately 1 in 12 cases of either disease. The expansion is translated to produce five dipeptide repeat proteins (DPRs) which aggregate in patient brain and are toxic in numerous models, though the mechanisms underlying this toxicity are poorly understood. Recent studies highlight nucleocytoplasmic transport impairments as a potential mechanism underlying neurodegeneration in C9orf72-linked disease, although the contribution of DPRs to this remains unclear. We expressed DPRs in HeLa cells, in the absence of repeat RNA. Crucially, we expressed DPRs at repeat-lengths found in patients (> 1000 units), ensuring our findings were relevant to disease. Immunofluorescence imaging was used to investigate the impact of each DPR on the nucleus, nucleocytoplasmic transport machinery and TDP-43 localisation. DPRs impaired the structural integrity of the nucleus, causing nuclear membrane disruption and misshapen nuclei. Ran and RanGAP, two proteins required for nucleocytoplasmic transport, were also mislocalised in DPR-expressing cells. Furthermore, DPRs triggered mislocalisation of TDP-43 to the cytoplasm, and this occurred in the same cells as Ran and RanGAP mislocalisation, suggesting a potential link between DPRs, nucleocytoplasmic transport impairments and TDP-43 pathology., (© 2022. The Author(s).)

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation., (© 2022. The Author(s).)

Clinical trials evaluating cardiac progenitor cells (CPC) demonstrated feasibility and safety, but no clear functional benefits. Therefore a deeper understanding of CPC biology is warranted to inform strategies capable to enhance their therapeutic potential. Here we have defined, using a label-free proteomic approach, the differential cytoplasmic and nuclear compartments of human CPC (hCPC). Global analysis of cytoplasmic repertoire in hCPC suggested an important hypoxia response capacity and active collagen metabolism. In addition, comparative analysis of the nuclear protein compartment identified a significant regulation of a small number of proteins in hCPC versus human mesenchymal stem cells (hMSC). Two proteins significantly upregulated in the hCPC nuclear compartment, IL1A and IMP3, showed also a parallel increase in mRNA expression in hCPC versus hMSC, and were studied further. IL1A, subjected to an important post-transcriptional regulation, was demonstrated to act as a dual-function cytokine with a plausible role in apoptosis regulation. The knockdown of the mRNA binding protein (IMP3) did not negatively impact hCPC viability, but reduced their proliferation and migration capacity. Analysis of a panel of putative candidate genes identified HMGA2 and PTPRF as IMP3 targets in hCPC. Therefore, they are potentially involved in hCPC proliferation/migration regulation., (© 2022. The Author(s).)

In a multi-branch family from Pakistan, individuals presenting with palmoplantar keratoderma segregate in autosomal dominant fashion, and individuals with intellectual disability (ID) segregate in apparent autosomal recessive fashion. Initial attempts to identify the ID locus using homozygosity-by-descent (HBD) mapping were unsuccessful. However, following an assumption of locus heterogeneity, a reiterative HBD approach in concert with whole exome sequencing (WES) was employed. We identified a known disease-linked mutation in the polymicrogyria gene, ADGRG1, in two affected members. In the remaining two (living) affected members, HBD mapping cross-referenced with WES data identified a single biallelic frameshifting variant in the gene encoding retinol dehydrogenase 14 (RDH14). Transcription data indicate that RDH14 is expressed in brain, but not in retina. Magnetic resonance imaging for the individuals with this RDH14 mutation show no signs of polymicrogyria, however cerebellar atrophy was a notable feature. RDH14 in HEK293 cells localized mainly in the nucleoplasm. Co-immunoprecipitation studies confirmed binding to the proton-activated chloride channel 1 (PACC1/TMEM206), which is greatly diminished by the mutation. Our studies suggest RDH14 as a candidate for autosomal recessive ID and cerebellar atrophy, implicating either disrupted retinoic acid signaling, or, through PACC1, disrupted chloride ion homeostasis in the brain as a putative disease mechanism., (© 2021. The Author(s).)

Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MB Thy1-scFv ) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging., (© 2021. The Author(s).)

Telomerases elongate the ends of chromosomes required for cell immortality through their reverse transcriptase activity. By using the model organism Saccharomyces cerevisiae we defined the order in which the holoenzyme matures. First, a longer precursor of the telomerase RNA, TLC1 is transcribed and exported into the cytoplasm, where it associates with the protecting Sm-ring, the Est and the Pop proteins. This partly matured telomerase is re-imported into the nucleus via Mtr10 and a novel TLC1-import factor, the karyopherin Cse1. Remarkably, while mutations in all known transport factors result in short telomere ends, mutation in CSE1 leads to the amplification of Y' elements in the terminal chromosome regions and thus elongated telomere ends. Cse1 does not only support TLC1 import, but also the Sm-ring stabilization on the RNA enableling Mtr10 contact and nuclear import. Thus, Sm-ring formation and import factor contact resembles a quality control step in the maturation process of the telomerase. The re-imported immature TLC1 is finally trimmed into the 1158 nucleotides long mature form via the nuclear exosome. TMG-capping of TLC1 finalizes maturation, leading to mature telomerase., (© 2021. The Author(s).)

Our lab has previously demonstrated Riluzole to be an effective drug in inhibiting proliferation and inducing apoptosis in both human and mouse osteosarcoma. Yes-associated protein is a transcription co-activator, known to be involved in cell proliferation or apoptosis depending on its protein partner. In the present study we investigated the role of YAP in apoptosis in osteosarcoma, we hypothesized that YAP may be activated by Riluzole to induce apoptosis in osteosarcoma. By knocking down the expression of YAP, we have demonstrated that Riluzole failed to induce apoptosis in YAP deficient osteosarcoma cells. Riluzole caused translocation of YAP from the cytoplasm to the nucleus, indicating YAP's role in apoptosis. Both Riluzole-induced phosphorylation of YAP at tyrosine 357 and Riluzole-induced apoptosis were blocked by inhibitors of c-Abl kinase. In addition, knockdown of c-Abl kinase prevented Riluzole-induced apoptosis in LM7 cells. We further demonstrated that Riluzole promoted interaction between YAP and p73, while c-Abl kinase inhibitors abolished the interaction. Subsequently, we demonstrated that Riluzole enhanced activity of the Bax promoter in a luciferase reporter assay and enhanced YAP/p73 binding on endogenous Bax promoter in a ChIP assay. Our data supports a novel mechanism in which Riluzole activates c-Abl kinase to regulate pro-apoptotic activity of YAP in osteosarcoma., (© 2021. The Author(s).)

Api5, is a known anti-apoptotic and nuclear protein that is responsible for inhibiting cell death in serum-starved conditions. The only known post-translational modification of Api5 is acetylation at lysine 251 (K251). K251 acetylation of Api5 is responsible for maintaining its stability while the de-acetylated form of Api5 is unstable. This study aimed to find out the enzymes regulating acetylation and deacetylation of Api5 and the effect of acetylation on its function. Our studies suggest that acetylation of Api5 at lysine 251 is mediated by p300 histone acetyltransferase while de-acetylation is carried out by HDAC1. Inhibition of acetylation by p300 leads to a reduction in Api5 levels while inhibition of deacetylation by HDAC1 results in increased levels of Api5. This dynamic switch between acetylation and deacetylation regulates the localisation of Api5 in the cell. This study also demonstrates that the regulation of acetylation and deacetylation of Api5 is an essential factor for the progression of the cell cycle., (© 2021. The Author(s).)

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations., (© 2021. The Author(s).)

Lipid rafts, sterol-rich and sphingolipid-rich microdomains on the plasma membrane are important in processes like cell signaling, adhesion, and protein and lipid transport. The virulence of many eukaryotic parasites is related to raft microdomains on the cell membrane. In the malaria parasite Plasmodium falciparum, glycosylphosphatidylinositol-anchored proteins, which are important for invasion and are possible targets for vaccine development, are localized in the raft. However, rafts are poorly understood. We used quick-freezing and freeze-fracture immuno-electron microscopy to examine the localization of monosialotetrahexosylganglioside (GM1) and monosialodihexosylganglioside (GM3), putative raft microdomain components in P. falciparum and infected erythrocytes. This method immobilizes molecules in situ, minimizing artifacts. GM3 was localized in the exoplasmic (EF) and cytoplasmic leaflets (PF) of the parasite and the parasitophorous vacuole (PV) membranes, but solely in the EF of the infected erythrocyte membrane, as in the case for uninfected erythrocytes. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) was localized solely in the PF of erythrocyte, parasite, and PV membranes. This is the first time that GM3, the major component of raft microdomains, was found in the PF of a biological membrane. The unique localization of raft microdomains may be due to P. falciparum lipid metabolism and its unique biological processes, like protein transport from the parasite to infected erythrocytes., (© 2021. The Author(s).)

A finite element analysis modelled diffusional generation of steady-state Ca 2+ microdomains within skeletal muscle transverse (T)-tubular-sarcoplasmic reticular (SR) junctions, sites of ryanodine receptor (RyR)-mediated SR Ca 2+ release. It used established quantifications of sarcomere and T-SR anatomy (radial diameter [Formula: see text]; axial distance [Formula: see text]). Its boundary SR Ca 2+ influx densities,[Formula: see text], reflected step impositions of influxes, [Formula: see text] deduced from previously measured Ca 2+ signals following muscle fibre depolarization. Predicted steady-state T-SR junctional edge [Ca 2+ ], [Ca 2+ ] edge, matched reported corresponding experimental cytosolic [Ca 2+ ] elevations given diffusional boundary efflux [Formula: see text] established cytosolic Ca 2+ diffusion coefficients [Formula: see text] and exit length [Formula: see text]. Dependences of predicted [Ca 2+ ] edge upon [Formula: see text] then matched those of experimental [Ca 2+ ] upon Ca 2+ release through their entire test voltage range. The resulting model consistently predicted elevated steady-state T-SR junctional ~ µM-[Ca 2+ ] elevations radially declining from maxima at the T-SR junction centre along the entire axial T-SR distance. These [Ca 2+ ] heterogeneities persisted through 10 4 - and fivefold, variations in D and w around, and fivefold reductions in d below, control values, and through reported resting muscle cytosolic [Ca 2+ ] values, whilst preserving the flux conservation ([Formula: see text] condition, [Formula: see text]. Skeletal muscle thus potentially forms physiologically significant ~ µM-[Ca 2+ ] T-SR microdomains that could regulate cytosolic and membrane signalling molecules including calmodulin and RyR, These findings directly fulfil recent experimental predictions invoking such Ca 2+ microdomains in observed regulatory effects upon Na + channel function, in a mechanism potentially occurring in similar restricted intracellular spaces in other cell types., (© 2021. The Author(s).)

Osteoblast differentiation leading to bone formation requires a coordinated transcriptional program. Osteoblastic cells with low level of microtubule actin crosslinking factor 1 (MACF1) show reduced osteoblast differentiation ability, however, the comprehensive mechanism of MACF1's action remains unexplored. In the current study, we found that MACF1 knockdown suppressed osteoblast differentiation by altering the transcriptome dynamics. We further identified two MACF1-interacted proteins, cyclin-dependent kinase 12 (CDK12) and MYST/Esa1-associated factor 6 (MEAF6), and two MACF1-interacted transcription factors (TFs), transcription factor 12 (TCF12) and E2F transcription factor 6 (E2F6), which repress osteoblast differentiation by altering the expression of osteogenic TFs and genes. Moreover, we found that MACF1 regulated cytoplasmic-nuclear localization of itself, TCF12 and E2F6 in a concentration-dependent manner. MACF1 oppositely regulates the expression of TCF12 and transcription factor 7 (TCF7), two TFs that drive osteoblast differentiation to opposite directions. This study reveals that MACF1, a cytoskeletal protein, acts as a sponge for repressors of osteoblast differentiation to promote osteoblast differentiation and contributes to a novel mechanistic insight of osteoblast differentiation and transcription dynamics.

Liquid-liquid phase separation is a major mechanism of subcellular compartmentalization 1,2 . Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism 3,4 , whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins 5-8 . We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs 9-11 suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.

Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3'UTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3' end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3' end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.

Mammary serine protease inhibitor (maspin) is a tumor suppressor gene that is downregulated during carcinogenesis and breast cancer progression. While the nuclear localization of maspin is essential for tumor suppression, we previously reported that the cytoplasmic localization of maspin was significantly correlated with poor prognosis in breast cancer patients. To understand the mechanisms that underlie oncogenic role of cytoplasmic maspin, we studied its biological function in breast cancer cell lines. Subcellular localization of maspin in MDA-MB-231 breast cancer cells was mainly detected in the cytoplasm, whereas in MCF10A mammary epithelial cells, maspin was present in both cytoplasm and nucleus. In MDA-MB-231 cells, maspin overexpression promoted cell proliferation and cell invasion, whereas maspin downregulation resulted in the opposite effect. Further, we observed that SRGN protein levels were increased in MDA-MB-231 cells stably overexpressing maspin. Finally, maspin overexpression in MDA-MB-231 cells resulted in the N-cadherin and epithelial mesenchymal transition (EMT)-related transcription factors upregulation, and TGFβ signaling pathway activation. These results suggested that cytoplasmic maspin enhances the invasive and metastatic potential in breast cancer cells with aggressive phenotype by inducing EMT via SRGN/TGFβ axis. This study demonstrated a novel biological function of cytoplasmic maspin in progression of breast cancer cells with an aggressive phenotype.

Transcytosis is a form of specialized transport through which an extracellular cargo is endocytosed, shuttled across the cytoplasm in membrane-bound vesicles, and secreted at a different plasma membrane surface. This important process allows membrane-impermeable macromolecules to pass through a cell and become accessible to adjacent cells and tissue compartments. Transcytosis also promotes redistribution of plasma membrane proteins and lipids to different regions of the cell surface. Here we review transcytosis and highlight in vivo studies showing how developing epithelial cells use it to change shape, to migrate, and to relocalize signaling molecules., (© 2021 The Authors.)

HBeAg, a non-particulate protein of hepatitis B virus (HBV), is translated from the precore/core region as a precursor, which is post-translationally modified. Subgenotype A1 of HBV, which is a risk factor for hepatocellular carcinoma (HCC), has unique molecular characteristics in the basic core promoter/precore regions. Carriers of A1 exhibit early HBeAg loss. We sought to further characterize the precore proteins of A1 in vitro. HuH-7 cells were transfected with subgenomic constructs expressing individual precore proteins. Western blot analysis using DAKO anti-core antibody showed the expected sizes and a 1 kDa larger band for P22, P20 and P17. Using confocal microscopy, a cytoplasmic accumulation of HBeAg and precursors was observed with P25-expressing plasmid, whereas P22 localized both in the cytoplasm and nucleus. P20 and P17, which lack the carboxy end of P22 showed strong nuclear accumulation, implicating a nuclear localization signal in the N-terminal 10 amino acids. G1862T, unique to subgenotype A1, is frequently found in HBV from HCC patients. P25 with G1862T showed delayed and reduced HBeAg expression/secretion. Knock-out of core in the replication competent clones led to precore protein accumulation in the cytoplasm/perinuclear region, and decreased HBeAg secretion. Knock-out of precore proteins increased HBsAg secretion but intracellular HBsAg expression was unaffected. Over-expression of precore proteins in trans led to decreased HBsAg expression and secretion. Intracellular trafficking of HBV A1 precore proteins was followed. This was unaffected by the CMV promoter and different cell types. In the viral context, precore protein expression was affected by absence of core, and affected HBsAg expression, suggesting an interrelationship between precore proteins, HBcAg and HBsAg. This modulatory role of HBeAg and its precursors may be important in viral persistence and ultimate development of HCC.

Early events of the human immunodeficiency virus 1 (HIV-1) lifecycle, such as post-entry virus trafficking, uncoating and nuclear import, are poorly characterized because of limited understanding of virus-host interactions. Here, we used mass spectrometry-based proteomics to delineate cellular binding partners of curved HIV-1 capsid lattices and identified Sec24C as an HIV-1 host dependency factor. Gene deletion and complementation in Jurkat cells revealed that Sec24C facilitates infection and markedly enhances HIV-1 spreading infection. Downregulation of Sec24C in HeLa cells substantially reduced HIV-1 core stability and adversely affected reverse transcription, nuclear import and infectivity. Live-cell microscopy showed that Sec24C co-trafficked with HIV-1 cores in the cytoplasm during virus ingress. Biochemical assays demonstrated that Sec24C directly and specifically interacted with hexameric capsid lattices. A 2.3-Å resolution crystal structure of Sec24C 228-242 in the complex with a capsid hexamer revealed that the Sec24C FG-motif bound to a pocket comprised of two adjoining capsid subunits. Combined with previous data 1-4 , our findings indicate that a capsid-binding FG-motif is conserved in unrelated proteins present in the cytoplasm (Sec24C), the nuclear pore (Nup153; refs. 3,4 ) and the nucleus (CPSF6; refs. 1,2 ). We propose that these virus-host interactions during HIV-1 trafficking across different cellular compartments are crucial for productive infection of target cells.

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

Scar formation as a result of corneal wound healing is a leading cause of blindness. It is a challenge to understand why scar formation is more likely to occur in the central part of the cornea as compared to the peripheral part. The purpose of this study was to unravel the underlying mechanisms. We applied RNA-seq to uncover the differences of expression profile in keratocytes in the central/peripheral part of the cornea. The relative quantity of mitochondrial RNA was measured by multiplex qPCR. The characterization of mitochondrial RNA in the cytoplasm was confirmed by immunofluoresence microscope and biochemical approach. Gene expression was analyzed by western blot and RT qPCR. We demonstrate that the occurrence of mitochondrial DNA common deletion is greater in keratocytes from the central cornea as compared to those of the peripheral part. The keratocytes with CD have elevated oxidative stress levels, which leads to the leakage of mitochondrial double-stranded RNA into the cytoplasm. The cytoplasmic mitochondrial double-stranded RNA is sensed by MDA5, which induces NF-κB activation. The NF-κB activation thereafter induces fibrosis-like extracellular matrix expressions and IL-8 mRNA transcription. These results provide a novel explanation of the different clinical outcome in different regions of the cornea during wound healing.

The genus Stentor is a relatively well-known ciliate owing to its lucid trumpet shape. Stentor pyriformis represents a green, short, and fat Stentor, but it is a little-known species. We investigated 124 ponds and wetlands in Japan and confirmed the presence of S. pyriformis at 23 locations. All these ponds were noticeably oligotrophic. With the improvement of oligotrophic culture conditions, we succeeded in long-term cultivation of three strains of S. pyriformis. The cytoplasm of S. piriformis contains a large number of 1-3 μm refractive granules that turn brown by Lugol's staining. The granules also show a typical Maltese-cross pattern by polarization microscopy, strongly suggesting that the granules are made of amylopectin-rich starch. By analyzing the algal rDNA, it was found that all S. pyriformis symbionts investigated in this study were Chlorella variabilis. This species is known as the symbiont of Paramecium bursaria and is physiologically specialized for endosymbiosis. Genetic discrepancies between C. variabilis of S. pyriformis and P. bursaria may indicate that algal sharing was an old incident. Having symbiotic algae and storing carbohydrate granules in the cytoplasm is considered a powerful strategy for this ciliate to withstand oligotrophic and cold winter environments in highland bogs.