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I have a clear memory of presenting my initial results about a “failed” protein at a lab meeting with my postdoctoral advisor Angus Lamond at the University of Dundee in Scotland and the rest of his group. It was the summer of 2000, and for the first few months of my postdoc I had been fusing green fluorescent protein with novel proteins that had recently been identified by mass spectrometry as residing in the nucleolus. I engineered HeLa cells to produce copious amounts of these fusion proteins, and watched where they went. Most migrated to the nucleolus, as expected, but one protein steadfastly refused. Instead, it formed nuclear dots that were much smaller than the large and obvious nucleoli.

I was really worried about the messiness of this result, but also intrigued. To my relief, instead of being disappointed that the protein was not doing what we had expected, Angus and my lab mates encouraged me to explore it further. The group had access to antibodies against many cellular structures, so I quickly established that these nuclear dots were different from any known nuclear bodies. But having generated much of my data with overexpressed protein, it was critical to make sure that the endogenous form of the protein also localized to the same nuclear dots, and that what I had seen were not simply artifacts of my approach.

GLOWING GREEN: Twenty years ago, GFP-labeled proteins in HeLa cells revealed the presence of the nuclear bodies now known as paraspeckles. (Human breast cancer cell from the MDA-MB-231 cell line shown here; scale bar is 5 μm.) Archa fox

We created an antibody against the protein and incubated it with HeLa cells. It was an incredibly nerve-wracking moment looking down the microscope to see what the antibody had stained. Thankfully, it worked. I was ecstatic when I saw that it had picked out the same small nuclear dots that I had identified with GFP. In 2002, I published a manuscript introducing the scientific community to paraspeckles— orbs of protein and nucleic acid 360 nanometers in diameter, squeezed next to the more famous and larger structures called nuclear speckles.

Since then, paraspeckles have become an established part of cell biology; there are more than 250 articles on them, and they have already found their way into some textbooks. We now know they are membraneless organelles seeded by a long noncoding RNA (lncRNA), formed through a well-characterized physical phenomenon known as liquid-liquid phase separation, and composed of numerous proteins and RNA molecules. We also know that they can alter gene regulation when cells get stressed, an important mechanism for maintaining cell homeostasis and one that appears to be disrupted in many diseases.

In 2006, I left the UK to establish my own paraspeckle lab in my home country of Australia. Reflecting on my postdoc reminds me of just how far we have come in such a short period of time. In addition to all we have learned about paraspeckle biology from in vitro work, many studies have now established that these structures appear in cells biopsied from human patients and from healthy mouse tissue samples. To have discovered a new cellular structure and have watched the birth of a research field focused on understanding that structure is an honor and a privilege. Looking ahead, I can see some big opportunities for paraspeckle biology, from using them as a model to understand lncRNAs and phase separation in the cell to developing therapeutics that modulate them in different diseases.

Discovering paraspeckles

When I joined Lamond’s lab in late 1999, I initially chose a project within my molecular biology expertise, cloning complementary DNAs for a bunch of proteins identified in the human nucleolus. But as I dove further into the problematic protein that wouldn’t localize to this known nuclear structure, I was quickly pulled out of my comfort zone. I had to learn new techniques to figure out why the protein was appearing in mass spectrometry analyses of nucleoli, but not in the nucleoli of my GFP-fusion–expressing cells.

I ended up adopting a technique that involved laser-bleaching the fluorescence of the wayward GFP-fusion protein. I then took many microscopic images over time to track where the bleached protein, which I named paraspeckle protein 1 (PSP1, subsequently renamed PSPC1), went in the cell. This method showed that it was travelling in and out of nucleoli under steady-state conditions, even though it was not substantially enriched within them. Mass spec was sensitive enough to pick up this trace of PSPC1 in the nucleoli that we could not see under the microscope.

To have discovered a new cellular structure and have watched the birth of a research field focused on understanding that structure is an honor and a privilege.

I later found that two other proteins—originally termed P54nrb and PSF, now called NONO and SFPQ, which are in the same family as PSPC1—were also enriched within paraspeckles. While PSPC1 was an unstudied protein at the time I named it, scientists knew that NONO and SFPQ played roles in gene regulation and RNA processing. It soon became obvious that RNA was a major component of paraspeckles, and various RNAs were integral to their formation. Treating cells with RNases made PSPC1, NONO, and SFPQ disappear from paraspeckles, showing it was RNA holding the proteins there. The picture emerged that paraspeckles were conglomerations of protein and RNA that were formed and maintained by what we now know as phase separation dynamics, a physical process that made them part of a group of cellular structures known as membraneless organelles.

See “These Organelles Have No Membranes”

I often saw paraspeckles in clusters on one side of the nucleus, and I could also see that only some of the HeLa cells’ micronuclei—small structures that carry fragments of chromosomes—had paraspeckles. Some nuclei must contain paraspeckle-forming DNA, while others did not, Angus and I thought; a particular part of the genome, maybe even a specific gene, might be causing paraspeckles to form. But the identity of that DNA was a mystery.

The period following my initial discovery in the early 2000s was a lonely time in paraspeckles research. Besides our group, there was only one other lab interested in paraspeckles—that of David Spector at Cold Spring Harbor Laboratory. A few years after our initial paraspeckle publication, independent work from his group found that NONO-bound RNA was retained in the nucleus, unable to enter the cytoplasm, where it could be translated into a protein. Then, in 2005, I received an email from Gérard Pierron at France’s National Center for Scientific Research (CNRS) near Paris.

Pierron wondered if paraspeckles might correspond to structures that his group had first observed with electron microscopy in the early 1990s. At the time, the researchers called the structures inter-chromatin granule associated zones. These were morphologically distinct from the rest of the nuclear material, or nucleoplasm, but did not have a molecular marker to distinguish them. I sent Pierron some antibody to PSPC1, which he tested against HeLa cells in his lab. Sure enough, he could see enrichment in the zones identified by electron microscopy, suggesting that these granules were indeed paraspeckles.

So actually, the French group had been the first to see and publish on what would later become known as paraspeckles—and suddenly I was not so alone.

Paraspeckle Formation These tiny subnuclear bodies typically measure 360 nanometers in diameter. They are composed of a long noncoding RNA (lncRNA) molecule called NEAT1, which serves as the seed. Proteins that bind to NEAT1 accumulate, self-associate, and recruit other proteins, forming a mature paraspeckle. Peg gerrity

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NEAT1, the long noncoding RNA of paraspeckles

In 2007, after I left Angus’s lab to start my own group at the University of Western Australia, I got another email about my paraspeckle work. Jeanne Lawrence, an epigenetics researcher based at the University of Massachusetts Medical School, requested some antibody to PSPC1. I happily shared my reagents, and then I did not think much more about it, until a few months later when Lawrence contacted me again to see if I was interested in a collaboration.

Her group was working on a lncRNA—broadly defined as any RNA of more than 200 nucleotides that does not appear to encode any protein—called NEAT1, and had used my PSPC1 antibody to show that the lncRNA colocalized with paraspeckles. When the lab knocked down NEAT1, paraspeckles could no longer form. I thought, here was the paraspeckle-forming gene that I had been searching for all these years.

Because NEAT1 is so long—more than 23,000 nucleotides—it is possible to label different parts of the RNA and see them appear in different zones of individual paraspeckles.

Together, our labs, along with Andrew Chess’s group at Icahn School of Medicine at Mount Sinai in New York City, assembled a narrative, published in 2009, of how NEAT1 seeds paraspeckle formation. Around the same time, two other groups—Spector’s Cold Spring Harbor lab and that of Tetsuro Hirose at Hokkaido University in Japan—reported similar results.

We had some inkling that other groups might be working on the same thing, but it still came as a surprise. It was unsettling to suddenly find myself in a competitive environment after working in relative obscurity, but the independent studies greatly strengthened the case for NEAT1 as a major component of paraspeckles. The findings also linked paraspeckles to the exciting world of lncRNAs at a time when the notion was just emerging that lncRNAs are functional, and not simply transcriptional noise. The debate over lncRNAs continues today. While there is a general acceptance that tens of thousands of lncRNAs are produced by the human genome, how many of these are functional is still controversial. NEAT1 has become an important model lncRNA with a clear cellular function: forming paraspeckles.

Because NEAT1 is so long—more than 23,000 nucleotides—it is possible to label different parts of the RNA and see them appear in different zones of individual paraspeckles. A paraspeckle is roughly spherical, with a shell and a core, as defined by the distinct protein and RNA composition of each of these regions. Using gold labelling and electron microscopy, Pierron’s group in Paris, in collaboration with us and others, showed that the 5' and 3' ends of NEAT1 are found in the shell, while the middle sequences of the RNA are found in the core. (See illustration on this page.) Another collaborative group that I was part of, led by Shinichi Nakagawa at Hokkaido University, later confirmed this with super-resolution imaging. Hirose’s group, working in concert with Nakagawa’s team, myself, and many others, found—by providing seed sequences for various paraspeckle-associated proteins to bind—that different regions of NEAT1 were required for directing this core-shell organization.

NEAT1 is now being finely dissected to understand how this one RNA can form a scaffold on which the tiny membraneless organelles can be built.

NEAT1’s role in the cell’s response to stress

Because NEAT1 is essential for paraspeckle formation, deleting NEAT1 in an animal makes a paraspeckle knockout. In 2011, Nakagawa established the NEAT1 knockout mouse. However, it showed no obvious phenotype. This was disappointing to me, and made it hard to justify continuing to work on paraspeckles. But as the Hokkaido-based team continued to scrutinize the mutants, it turned out that there was a phenotype: some female knockout mice had reduced fertility. Nakagawa found that paraspeckles are abundant in the corpus luteum that forms in the ovary and emits progesterone after the release of an ovum. Loss of NEAT1 prevented the corpus luteum from forming in some, but not all, of the knockout females.

That some animals appeared to be more reliant on paraspeckles than others hinted at the possibility that environmental factors are at play when it comes to paraspeckle function. Sure enough, my group and others have since found that various stressors can trigger the formation of abundant, larger-than-normal paraspeckles that sometimes take on an oblong shape. Paraspeckles seem to be part of the cell stress response.

NUCLEAR SPOTS: A HeLa cell nucleus with DNA stained blue and paraspeckle markers in green and red. The large circles within the nucleus are nucleoli. Scale bar is 5 μm. ARCHA FOX

The key is NEAT1 transcription: more NEAT1 RNA means more paraspeckles. A 2018 genome-wide screen conducted by Lingling Chen at the Shanghai Institute of Biochemistry and Cell Biology identified more than 100 factors that increase NEAT1 transcription. These include molecular signals of mitochondrial disturbance, a driver of many diseases.

See “Power Failure”

As paraspeckle numbers increase within a cell due to stress, they sequester paraspeckle-associated transcription factors such as SFPQ—one of the first two proteins I discovered to be a component of paraspeckles, along with PSPC1—and this changes expression of the downstream target genes. Large paraspeckles, which can grow to be up to 2 micrometers long, also sequester specific mRNAs, stopping them from being exported from the nucleus and translated in the cytoplasm. (See illustration on page 37.) This paraspeckle-driven gene regulation is important in immune responses to many types of infection, and it helps cells when mitochondria get stressed by regulating mRNAs that encode key mitochondrial proteins.

The story that has emerged is that paraspeckles act as buffers when cells are stressed, helping them maintain homeostasis and avoid apoptosis. This function is likely useful for long-lived cells such as neurons, but also may play a role in some types of tumor formation. The role of paraspeckles in cancer is still an active area of study, however, and thus far there are conflicting data in different cancer types. While one big study found that paraspeckles may be oncogenic, another paper found that the structures can actually suppress cancer. Nevertheless, it appears that paraspeckles can be both good and bad when it comes to disease.

Paraspeckle Function When a cell is stressed, various triggers can cause it to increase the production of the lncRNA NEAT1, leading to the formation of more paraspeckles. These bodies can grow to up to 2 micrometers in length, changing from a spherical shape to oblong and sometimes branched structures. They trap various proteins and mRNAs, hindering their function and thereby affecting the cell’s continued response to stress. PEG GERRITY 1 As they accumulate proteins and RNAs, paraspeckles can become linked together, growing bigger, oblong, and sometimes branched. 2 Greater abundance of NEAT1 leads to more paraspeckles. 3 Specific messenger RNAs become trapped in paraspeckles and cannot reach the cytoplasm for translation. 4 Paraspeckles act as a sponge, soaking up the proteins from the nucleoplasm. 5 Paraspeckles trap gene-regulating proteins, depleting them from target sites on the genome and thereby altering transcription.



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Getting in phase with disorder

Even with all the excitement about the NEAT1 RNA, I never lost focus on the paraspeckle proteins that are also needed for these little structures to form. In 2012, Hirose and colleagues published a landmark paper expanding the paraspeckle proteome from the three I originally found up to a total of 40 different protein types.

My postdoc Sven Hennig and I teamed up with Hirose to interrogate the interactions among these molecules and found that some parts of the proteins with no predicted structure, called low complexity domains, were very important. In collaboration with Charlie Bond, also at the University of Western Australia, we showed that the low complexity domains of the paraspeckle proteins formed hydrogels, jelly-like globs that are neither liquid nor solid, implicating these proteins in the phenomenon of liquid-liquid phase separation (LLPS). LLPS is a hot new area in cell biology, and it may eventually explain how cellular structures and macromolecular complexes form without membranes to compartmentalize the cytoplasm and nucleus.

In the case of paraspeckles, after binding to NEAT1, proteins self-associate and recruit other paraspeckle proteins that glue RNA-protein particles into a mature paraspeckle. While the material properties of paraspeckles are still being worked out, I anticipate that our wealth of knowledge about their composition and organization will make them highly attractive as a model membraneless organelle to help scientists gain new ground in continuing research on phase separation.

My hope for paraspeckle biology is that we will one day have a complete molecular model of a paraspeckle, down to the atomic level, with a full suite of molecular tools to break it down and build it up. With a better understanding of why cells make paraspeckles, especially when stressed, we could in theory use these tools therapeutically to modulate the structures in different diseases. Although we have learned a lot since the discovery of paraspeckles nearly two decades ago due to the hard work and creativity of the members of my and other research groups, there is undoubtedly much more to understand. I look forward to being part of that adventure.

Archa Fox is an associate professor and Australian Research Council Future Fellow at the University of Western Australia and an affiliate with the Harry Perkins Institute of Medical Research.