ComBio special: The peculiar world of the paraspeckle

Deep within the nucleus of the cell, lurking amongst various Cajal bodies, PML bodies and the nucleolus itself, lie strange little compartments called paraspeckles. Often found in proximity to the better known splicing speckles, paraspeckles were only discovered five years ago and their true role in the cell is still a matter of debate.

Dr Archa Fox has a good idea of the main function of the paraspeckle, however, and she should know, having discovered them in the first place. Fox believes paraspeckles are involved in post-transcriptional control of gene expression and are responsible for regulating certain RNAs in a most peculiar manner.

At the moment, it is known that paraspeckles are responsible for the nuclear retention of one type of murine RNA that has a role in the nitrous oxide stress response pathway, but Fox suspects they may have a role in much more than that.

Fox first described the existence of paraspeckles in Current Biology while a postdoctoral fellow in 2002, and named them with the assistance of her supervisor at the University of Dundee, Professor Angus Lamond.

"I remember we were both standing in the corridor thinking about what on earth we were going to call these things," Fox says. "We decided to call them paraspeckles because they are often found in close conjunction with splicing speckles [nuclear domains thought to store mRNA splicing factors]. They are in a similar place but alongside or parallel to splicing speckles - so 'para speckle'."

Fox, now back in Australia running her own lab at the Western Australian Institute of Medical Research (WAIMR) in Perth after six and a half years in Scotland, says that while paraspeckles may have been named with splicing speckles in mind, they are quite different beasts.

"And unlike splicing speckles, which have been known for a long time now but we still don't have a clear idea as to their function, we are getting closer to what the actual function of the paraspeckle is, which is really gratifying."

Fine molecular detail

Fox, who will discuss her work in the RNA-protein function stream at ComBio next week, became interested in nuclear organisation when she become the first PhD student in Professor Merlin Crossley's new lab at the University of Sydney. Crossley had just returned from the US to start up his own lab to further study gene regulation, and it was here that Fox says the tone was set for her subsequent career.

At Crossley's lab Fox began work on the interaction between two zinc finger transcription factors - GATA-1 and FOG-1. The lab specialised in haematopoietic diseases and the transcription factors involved in blood cell differentiation.

It was only after her PhD was completed and she had moved on that a link between her work and disease was published, "but the work showed that with one particular mutation associated with disease, you could instantly go back and say that mutation altered that GATA-FOG interaction, which was fascinating for me," she says. "You can look at the fine details and they do actually all add up to the bigger picture."

Fox has now moved from that sphere but the theme is still the same - uncovering the big questions about gene expression and how genes are turned on and turned off. "That has been the theme of my career so far, even though I'm approaching it from a completely different area now."

That different mode of investigation was initiated when Fox won a Wellcome Trust International Fellowship - and assorted others - which allowed her six years at the University of Dundee. In the new lab, the focus was more on nuclear organisation.

"Previously I was looking at individual transcription factors, turning genes on and off at the DNA level, but this lab was taking a more global view of the nucleus as a whole. It gave me a cell biology perspective, about how the actual structure and organisation of the nucleus affects gene expression."

Fox points out that as organisms become more complex, so to does their nuclear organisation, from bacteria that exist without a nucleus to the simply organised nuclei of yeast and mammals, with their extraordinarily complicated internal structures.

Nuclear organisation has been studied for decades but it is only the advent of green fluorescent protein (GFP) and its ability to label the different structures within the cell, as well as high-resolution microscopy, which have allowed scientists to delve into what Fox calls the "fine molecular detail".

Post-transcriptional control

The lab at Dundee took a number of approaches to tackling the problems of understanding the nucleus. One was a proteomic approach, with Fox initially working on sub-nuclear particle purification and the proteomic identification of their components.

She initially worked on the proteome of the nucleolus, the biggest, densest part of the nucleus where the ribosomes are made. From there she tracked down the paraspeckle and has been studying it ever since.

"We think they are involved in post-transcriptional control of gene expression, so we are talking more about the RNA world," she says. "And RNA-protein interaction is really the basis.

"A lot of the basic nuclear organisation revolves around processing all of the new RNA that is being made - splicing, capping and facilitating the export of the RNA out of the nucleus. A lot of the actual organisation of the nucleus is dealing with that. We know for example that when you stop transcription and you stop production of RNA, a lot of the organisation of the nucleus changes and reorganises.

"Of course there is obviously DNA replication, DNA repair and transcription and a lot of other things going on in the nucleus as well, but we think the paraspeckles are involved in that post-transcriptional level."

Fox says the proteins found within paraspeckles are all RNA binding proteins. Current thinking is that it is the specific interactions with RNA binding proteins and the target RNAs that are key to building the paraspeckles, maintaining them and their function, she says.

While cytoplasmic organelles have membranes to enclose their contents, sub-nuclear structures do not, and yet there is a definite organisational structure.

"All of that organisation is mediated by protein-protein interaction, protein-RNA or protein-DNA interactions and only those interactions that hold everything together. That's why one of my main research focuses is trying to dissect the fine details of these molecular interactions to try to understand how these things are formed and how they are doing their job."

Nuclear retention

In 2005, a team led by David Spector from the Cold Spring Harbor Laboratory in the US published a study that validated Fox's theories. The study, 'Regulating Gene Expression through RNA Nuclear Retention', published in Cell in October of that year, found that a particular RNA, rather than leaving the nucleus to do its work in the cytoplasm, was actually trapped within the nucleus, being built up and enriched, contrary to normal behaviour.

This RNA, called CAT2 transcribed nuclear RNA (CTN-RNA), is encoded by the mouse cationic amino acid transporter 2 (mCAT2) gene and is responsible for a membrane protein that is involved in the uptake of cationic amino acids, believed to be involved in the nitrous oxide stress response pathway.

"There have been lots of studies visualising and tagging generic RNA molecules and showing that they get made and diffuse out of the nucleus as quickly as they can," Fox says.

"But this is a different one. What was happening was that the RNA was getting trapped in the paraspeckle, being bound by the specific paraspeckle proteins and being held there. What they then showed is that when the cell received a particular signal, that signal was somehow transmitted into the nucleus and the RNA was released from the paraspeckles and then allowed to go the cytoplasm and made into a protein.

"The hypothesis is that it's another level of control - that's what makes us the complex and unique organisms that we are. Like microRNAs and other non-coding RNAs, this is another facet. It seems to be a way that the cell uses to more tightly control these particular RNAs.

"It's thought that for this particular gene, (the process) is saving the cell about 25 minutes; the fact that it doesn't have to transcribe the gene, it doesn't have to splice it or process it, the RNA is just sitting there.

"It is then released in a rapid response mechanism - it's not as rapid as say a phosphorylation event, where you have an inactive protein, suddenly phosphorylate it and it's active - but it's more rapid than requiring a transcription factor to go into the nucleus, transcribe a gene, splice it and cap it. The hypothesis is that this thing is being made in order to facilitate a quick response to stress."

Fox says that currently, mCAT2 is the only gene known that undergoes this process, but she suspects from other work that there are likely to be many more genes regulated in this manner.

"That one is mouse specific and yet we see paraspeckles in human cells and lots of other different cell types. We know that the proteins are the same, so we think there will be a slew of RNAs that are regulated by this.

"We don't know what they are yet so that's one of my main projects, to try to find out what they are, and to find out if nuclear retention at paraspeckles is involved in development or differentiation and cancer.

"We want to know if cancer cells are hijacking this mechanism to regulate some of the key genes for cancer in this way. If you can save that time, and if you have genes that are being regulated for use in that stress response, you would have an advantage as a cancer cell by escaping the normal controls."