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 RNAscaffolded 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.

Long noncoding RNAs (lncRNAs) can act as tumour suppressor or oncogenes to contrast/promote tumour cell proliferation via RNA-dependent mechanisms. Recently, genome sequencing has identified elevated densities of tumour somatic single nucleotide variants (SNVs) in lncRNA genes. However, this has been attributed to phenotypically-neutral “passenger” processes, and the existence of positively-selected fitness-altering “driver” SNVs acting via lncRNAs has not been addressed. We developed and used ExInAtor2, an improved driver-discovery pipeline, to map pancancer and cancer-specific mutated lncRNAs across an extensive cohort of 2583 primary and 3527 metastatic tumours. The 54 resulting lncRNAs are mostly linked to cancer for the first time. Their significance is supported by a range of clinical and genomic evidence, and display oncogenic potential when experimentally expressed in matched tumour models. Our results revealed a striking SNV hotspot in the iconic NEAT1 oncogene, which was ascribed by previous studies to passenger processes. To directly evaluate the functional significance of NEAT1 SNVs, we used in cellulo mutagenesis to introduce tumour-like mutations in the gene and observed a consequent increase in cell proliferation in both transformed and normal backgrounds. Mechanistic analyses revealed that SNVs alter NEAT1 ribonucleoprotein assembly and boost subnuclear paraspeckles. This is the first experimental evidence that mutated lncRNAs can contribute to the pathological fitness of tumour cells.

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.

Tumour DNA contains thousands of single nucleotide variants (SNVs) in non-protein-coding regions, yet it remains unclear which are “driver mutations” that promote cell fitness. Amongst the most highly mutated non-coding elements are long noncoding RNAs (lncRNAs), which can promote cancer and may be targeted therapeutically. We here searched for evidence that driver mutations may act through alteration of lncRNA function. Using an integrative driver discovery algorithm, we analysed single nucleotide variants (SNVs) from 2583 primary tumours and 3527 metastases to reveal 54 candidate “driver lncRNAs” (FDR<0.1). Their relevance is supported by enrichment for previously-reported cancer genes and by clinical and genomic features. Using knockdown and transgene overexpression, we show that tumour SNVs in two novel lncRNAs can boost cell fitness. Researchers have noted particularly high yet unexplained mutation rates in the iconic cancer lncRNA, NEAT1. We apply in cellulo mutagenesis by CRISPR-Cas9 to identify vulnerable regions of NEAT1 where SNVs reproducibly increase cell fitness in both transformed and normal backgrounds. In particular, mutations in the 5’ region of NEAT1 alter ribonucleoprotein assembly and boost the population of subnuclear paraspeckles. Together, this work reveals function-altering somatic lncRNA mutations as a new route to enhanced cell fitness during transformation and metastasis.