Cold Spring Harbor, N.Y. - Most of the DNA in the nucleus of each of our cells is converted into RNA, but only a small fraction of these RNA molecules serve as coding templates for the synthesis of proteins. Of the remaining RNAs, known as "non-coding" RNAs (ncRNA), the functions of a scant few are known: they inhibit the activity of genes or modify them by altering the way in which DNA is packaged within cells. What the rest of them do within cells is largely a mystery.
Professor David L. Spector, Ph.D., and a team led by graduate student Hongjae Sunwoo at Cold Spring Harbor Laboratory (CSHL), have expanded our knowledge of ncRNA functions by uncovering a unique structure-building role for two ncRNA molecules. In a paper published in the March 1st issue of Genome Research, they show that ncRNAs called MENε and MENβ organize and maintain the structure of paraspeckles, a compartment within the cell's nucleus.
RNAs as structural components
Unlike its counterparts in simple organisms like yeast, the nucleus in mammalian cells has an extraordinarily complicated internal structure. In addition to the DNA-protein complex known as chromatin, the nucleus is organized with compartments such as the nucleolus, PML bodies, Cajal bodies, and many others. Cell biologists have long wondered how these compartments are organized, knowing only that each has a definite and precise form despite not being bound and contained within a membrane.
"The idea that some of these structures might somehow be supported by RNA molecules first surfaced in studies in the 1970s," according to Professor Spector. His lab found further evidence for this idea in 2005 when they showed that paraspeckles - each nucleus has about 10 to 30 of these scattered around - fell apart when cells were treated with an enzyme that destroys RNA. "But," Spector says, "a specific RNA molecule that gives paraspeckles their structural integrity was never found."
Spector, who is an avid explorer of ncRNAs and their activities in cells, thought ncRNAs were a good candidate for such a role, especially since many of them stay in the nucleus instead of getting expelled into the cell's cytoplasm like their protein-coding RNA cousins.
ncRNAs localize to paraspeckles
Cells usually increase their production of ncRNAs when they ramp up their metabolic activity - for example, when they differentiate during development. For this reason, Spector's team, in collaboration with John Mattick's laboratory (Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia), hunted for their ncRNA quarry in a type of stem cell that differentiates into muscle fibers. They narrowed their focus to 14 confirmed ncRNAs, each of which was produced at levels that were at least two times higher or lower in the differentiated cells.
"One way to figure out hints as to what ncRNAs do is to find out where they localize within cells, which might reveal the molecules they interact with and the pathways they participate in," explains Spector. Using a technique that uses a fluorescence signal to tag molecules within cells, Spector's team traced two of their 14 ncRNAs to paraspeckles, tiny compartments that look like dense dots anchored within the nucleus.
MENε and MENβ are structural organizers
The paraspeckles were larger in differentiated muscle cells than in their stem-cell precursors - a sign that their structure might depend on ncRNAs, whose levels are also higher in muscle cells. The team proved this point by inserting into the cells molecules that bind to these ncRNAs and trigger their destruction. When these specific ncRNAs were depleted in this way, the paraspeckles disappeared. In addition, when the scientists depleted the levels of the ncRNAs and blocked the production of new ncRNA molecules, paraspeckles failed to form suggesting that the ncRNAs were essential to both initiate and maintain these nuclear structures.
Thus, not only did MENε and MENβ appear to maintain paraspeckle structure; they were also critical for the assembly of new paraspeckles. Spector's team also found that MENε and MENβ latch on to a paraspeckle protein called Nono. But how and whether this interaction is related to paraspeckle structure remains to be worked out.
The function of ncRNA-built paraspeckles
Scientists are still unsure about what paraspeckles do within cells. When they were discovered in 2002, it was believed they were involved in controlling gene activity "post-transcriptionally," i.e., after a gene's DNA has been converted into RNA.
One such post-transcriptional control mechanism was uncovered in 2005 by Spector's group, who showed that paraspeckles were warehouses for storing a specific RNA molecule. Only when the cell needed to respond to stress signals was this molecule processed and released into the cytoplasm, where it was used to synthesize protein.
Scientists estimate that storing pre-made RNAs in the paraspeckles and releasing them during times of need speeds the cell's response to stress by about 25 minutes, in certain cases, and bypasses the cell's need to prepare the RNA from scratch.
"Paraspeckles might be part of the cell's rapid response mechanism to stress," says Spector. "It might allow cells to meet challenges such as viral infection more quickly." Experiments performed by others bear him out: levels of paraspeckle-building MEN RNAs have been observed to increase in mouse brain cells infected by the Rabies virus.
Spector's work on nuclear ncRNAs continues. "Whether other nuclear compartments also similarly serve as RNA stores and what roles ncRNAs might play in nuclear structure and function are questions that will keep us busy for a while," he says.
"MEN ε/β nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles," was published advance of print on December 22nd, 2008 and appears in the March 1st print issue of Genome Research. The full citation is: Hongjae Sunwoo, Marcel E. Dinger, Jeremy E. Wilusz, Paulo P. Amaral, John S. Mattick, and David L. Spector. The paper can be found online at http://genome.
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