Tumour mutations in long noncoding RNAs enhance cell fitness.

Long noncoding RNAs (lncRNAs) are linked to cancer via pathogenic changes in their expression levels. Yet, it remains unclear whether lncRNAs can also impact tumour cell fitness via function-altering somatic "driver" mutations. To search for such driver-lncRNAs, we here perform a genome-wide analysis of fitness-altering single nucleotide variants (SNVs) across a cohort of 2583 primary and 3527 metastatic tumours. The resulting 54 mutated and positively-selected lncRNAs are significantly enriched for previously-reported cancer genes and a range of clinical and genomic features. A number of these lncRNAs promote tumour cell proliferation when overexpressed in in vitro models. Our results also highlight a dense SNV hotspot in the widely-studied NEAT1 oncogene. To directly evaluate the functional significance of NEAT1 SNVs, we use in cellulo mutagenesis to introduce tumour-like mutations in the gene and observe a significant and reproducible increase in cell fitness, both in vitro and in a mouse model. Mechanistic studies reveal that SNVs remodel the NEAT1 ribonucleoprotein and boost subnuclear paraspeckles. In summary, this work demonstrates the utility of driver analysis for mapping cancer-promoting lncRNAs, and provides experimental evidence that somatic mutations can act through lncRNAs to enhance pathological cancer cell fitness.

Confined environments induce polarized paraspeckle condensates.

Cancer cells experience confinement as they navigate the tumour microenvironment during metastasis. Recent studies have revealed that the nucleus can function as a 'ruler' for measuring physical confinement via membrane tension, allowing for compression-sensitive changes in migration. Cell nuclei contain many nuclear bodies that form when their components phase separate and condense within permissive local regions within the nucleus. However, how sub-nuclear organisation and phase separation changes with cell confinement and compression is largely unknown. Here we focus on paraspeckles, stress-responsive subnuclear bodies that form by phase separation around the long non-coding RNA NEAT1. As cells entered moderate confinement, a significant increase in paraspeckle number and size was observed compared to unconfined cells. Paraspeckle polarization bias towards the leading edge was also observed in confinement, correlating with regions of euchromatin. Increasing paraspeckle abundance resulted in increases in confined migration likelihood, speed, and directionality, as well as an enhancement of paraspeckle polarization towards the leading edge. This polarization of paraspeckle condensates may play a key role in regulating confined migration and invasion in cancer cells, and illustrates the utility of microchannel-based assays for identifying phenomena not observed on 2D or 3D bulk substrates.

NONO enhances mRNA processing of super-enhancer-associated GATA2 and HAND2 genes in neuroblastoma.

High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.

Single-Cell Transcriptome Analysis Reveals Paraspeckles Expression in Osteosarcoma Tissues.

Nuclear paraspeckles are subnuclear bodies contracted by nuclear-enriched abundant transcript 1 (NEAT1) long non-coding RNA, localised in the interchromatin space of mammalian cell nuclei. Paraspeckles have been critically involved in tumour progression, metastasis and chemoresistance. To this date, there are limited findings to suggest that paraspeckles, NEAT1 and heterogeneous nuclear ribonucleoproteins (hnRNPs) directly or indirectly play roles in osteosarcoma progression. Herein, we analysed NEAT1, paraspeckle proteins (SFPQ, PSPC1 and NONO) and hnRNP members (HNRNPK, HNRNPM, HNRNPR and HNRNPD) gene expression in 6 osteosarcoma tumour tissues using the single-cell RNA-sequencing method. The normalised data highlighted that the paraspeckles transcripts were highly abundant in osteoblastic OS cells, except NEAT1, which was highly expressed in myeloid cell 1 and 2 subpopulations.

The Long and the Short of It: NEAT1 and Cancer Cell Metabolism.

The long noncoding RNA NEAT1 is known to be heavily dysregulated in many cancers. A single exon gene produces two isoforms, NEAT1_1 and NEAT1_2, through alternative 3'-end processing. As the longer isoform, NEAT1_2 is an essential scaffold for nuclear paraspeckle formation. It was previously thought that the short NEAT1_1 isoform only exists to keep the NEAT1 locus active for rapid paraspeckle formation. However, a recent glycolysis-enhancing function for NEAT1_1, contributing to cancer cell proliferation and the Warburg effect, has been demonstrated. Previous studies have mainly focused on quantifying total NEAT1 and NEAT1_2 expression levels. However, in light of the NEAT1_1 role in cancer cell metabolism, the contribution from specific NEAT1 isoforms is no longer clear. Here, the roles of NEAT1_1 and NEAT1_2 in metabolism and cancer progression are discussed.

Species-specific formation of paraspeckles in intestinal epithelium revealed by characterization of NEAT1 in naked mole-rat.

Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.

The role of G-Quadruplex DNA in Paraspeckle formation in cancer.

Paraspeckles are RNA-protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

Caveolin-1-driven membrane remodelling regulates hnRNPK-mediated exosomal microRNA sorting in cancer.

BACKGROUND: Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non-caveolar caveolin-1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1-induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis-promoting microRNAs. RESULTS: We identify hnRNPK as a CAV1-regulated microRNA binding protein. In the absence of CAVIN1, non-caveolar CAV1 drives localisation of hnRPNK to multi-vesicular bodies (MVBs), recruiting AsUGnA motif-containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl-ß-cyclodextrin or by treatment with n-3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer. CONCLUSIONS: Taken together, these results support a novel pan-cancer mechanism for CAV1-driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment.

Paraspeckle nuclear condensates: Global sensors of cell stress?

Paraspeckles are nuclear condensates, or membranelees organelles, that are built on the long noncoding RNA, NEAT1, and have been linked to many diseases. Although originally described as constitutive structures, here, in reviewing this field, we develop the hypothesis that cells increase paraspeckle abundance as part of a general stress response, to aid pro-survival pathways. Paraspeckles increase in many scenarios: when cells transform from one state to another, become infected with viruses and bacteria, begin to degenerate, under inflammation, in aging, and in cancer. Cells increase paraspeckles by increasing transcription of NEAT1 and adjusting its RNA processing. These increases in NEAT1 are driven by numerous stress-sensing signaling pathways, including signaling to mitochondria and stress granules, revealing crosstalk between the cytoplasm and nucleoplasm in the stress response. Thus, paraspeckles are an important piece of the puzzle in cellular homeostasis, and could be considered RNA-scaffolded nuclear equivalents of dynamic stress-induced structures that form in the cytoplasm. We speculate that, in general, cells rely on phase-separated paraspeckles to transiently tweak gene regulation in times of cellular flux.

NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma.

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

Matrix stiffness-sensitive long noncoding RNA NEAT1 seeded paraspeckles in cancer cells.

Cancer progression is influenced by changes in the tumor microenvironment, such as the stiffening of the extracellular matrix. Yet our understanding of how cancer cells sense and convert mechanical stimuli into biochemical signals and physiological responses is still limited. The long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), which forms the backbone of subnuclear "paraspeckle" bodies, has been identified as a key genetic regulator in numerous cancers. Here, we investigated whether paraspeckles, as defined by NEAT1 localization, are mechanosensitive. Using tunable polyacrylamide hydrogels of extreme stiffnesses, we measured paraspeckle parameters in several cancer cell lines and observed an increase in paraspeckles in cells cultured on soft (3 kPa) hydrogels compared with stiffer (40 kPa) hydrogels. This response to soft substrate is erased when cells are first conditioned on stiff substrate, and then transferred onto soft hydrogels, suggestive of mechanomemory upstream of paraspeckle regulation. We also examined some well-characterized mechanosensitive markers, but found that lamin A expression, as well as YAP and MRTF-A nuclear translocation did not show consistent trends between stiffnesses, despite all cell types having increased migration, nuclear, and cell area on stiffer hydrogels. We thus propose that paraspeckles may prove of use as mechanosensors in cancer mechanobiology.

The emerging roles of hnRNPK.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an DNA/RNA-binding protein and regulates a wide range of biological processes and disease pathogenesis. It contains 3 K-homologous (KH) domains, which are conserved in other RNA-binding proteins, mediate nucleic acid binding activity, and function as an enhancer or repressor of gene transcription. Phosphorylation of the protein alters its regulatory function, which also enables the protein to serve as a docking platform for the signal transduction proteins. In terms of the function of hnRNPK, it is central to many cellular events, including long noncoding RNA (lncRNA) regulation, cancer development and bone homoeostasis. Many studies have identified hnRNPK as an oncogene, where it is overexpressed in cancer tissues compared with the nonneoplastic tissues and its expression level is related to the prognosis of different types of host malignancies. However, hnRNPK has also been identified as a tumour suppressor, as it is important for the activation of the p53/p21 pathway. Recently, the protein is also found to be exclusively related to the regulation of paraspeckles and lncRNAs such as Neat1, Lncenc1 and Xist. Interestingly, hnRNPK has been found to associate with the Kabuki-like syndrome and Au-Kline syndrome with prominent skeletal abnormalities. In vitro study revealed that the hnRNPK protein is essential for the formation of osteoclast, in line with its importance in the skeletal system.

ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles.

Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.

NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length.

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins.

Members of the Drosophila behavior human splicing (DBHS) protein family are nuclear proteins implicated in many layers of nuclear functions, including RNA biogenesis as well as DNA repair. Definitive of the DBHS protein family, the conserved DBHS domain provides a dimerization platform that is critical for the structural integrity and function of these proteins. The three human DBHS proteins, splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and non-POU domain-containing octamer-binding protein (NONO), form either homo- or heterodimers; however, the relative affinity and mechanistic details of preferential heterodimerization are yet to be deciphered. Here we report the crystal structure of a SFPQ/PSPC1 heterodimer to 2.3-Å resolution and analyzed the subtle structural differences between the SFPQ/PSPC1 heterodimer and the previously characterized SFPQ homodimer. Analytical ultracentrifugation to estimate the dimerization equilibrium of the SFPQ-containing dimers revealed that the SFPQ-containing dimers dissociate at low micromolar concentrations and that the heterodimers have higher affinities than the homodimer. Moreover, we observed that the apparent dissociation constant for the SFPQ/PSPC1 heterodimer was over 6-fold lower than that of the SFPQ/NONO heterodimer. We propose that these differences in dimerization affinity may represent a potential mechanism by which PSPC1 at a lower relative cellular abundance can outcompete NONO to heterodimerize with SFPQ.

Nuclear bodies: news insights into structure and function.

The cell nucleus contains a number of different dynamic bodies that are variously composed of proteins and generally, but not always, specific RNA molecules. Recent studies have revealed new understanding about nuclear body formation and function in different aspects of nuclear metabolism. Here, we focus on findings describing the role of nuclear bodies in the biogenesis of specific ribonucleoprotein complexes, processing of key mRNAs, and subnuclear sequestration of protein factors. We highlight how nuclear bodies are involved in stress responses, innate immunity and tumorigenesis. We further review organization of nuclear bodies and principles that govern their assembly, highlighting the pivotal role of scaffolding noncoding RNAs, and liquid-liquid phase separation, which are transforming our picture of nuclear body formation.

Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development.

Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process. VIDEO ABSTRACT.

Functional dissection of NEAT1 using genome editing reveals substantial localization of the NEAT1_1 isoform outside paraspeckles.

Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed "microspeckles," which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.