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Jul 9

Research Byte: Packard scientists identify RNA structure critical for the assembly of membraneless organelles

Packard Center News, Research Bytes
In a new study, Packard scientist Jiou Wang and colleagues showed that RNA structures called G-quadruplexes play a role in the formation and function of a type of granule called a paraspeckle.

As scientists have delved deeper into the molecular biology of ALS, one of the processes that continue to emerge is a disturbance to the dynamics of membraneless organelles--bodies of concentrated RNA and protein. Unlike membrane-bound organelles, these bodies or granules are not surrounded by a membrane but held together by biochemical and physical interactions between the RNA and protein building blocks. In previous studies, scientists have linked abnormal functioning of membraneless organelles to age-related neurodegenerative diseases like ALS. In a new study in Nucleic Acids Research, Packard scientist Jiou Wang, a neuroscientist at Johns Hopkins University, and colleagues showed that RNA structures called G-quadruplexes play a role in the formation and function of a type of granule called a paraspeckle.

Paraspeckles are found in the nucleus and are believed to help control gene expression through diverse pathways. They play a role in a variety of physiological processes, including those in the nervous system and in fertility in females. The paraspeckles themselves are built on a physical scaffolding of a long non-coding RNA (lncRNA) called NEAT1. Each paraspeckle consists of approximately 50 NEAT1 ribonucleoprotein particles. But scientists didn’t know what mediated the interactions between NEAT1 lncRNA and its key protein partners, although some researchers hypothesized that three-dimensional structures could play a role.

Wang and colleagues thought that one potential candidate for this structure is G-quadruplex, which is a four-stranded structure formed by guanine-rich RNA. Although G-quadruplexes are known to play a role in numerous basic biological processes, they are especially important in ALS since the repeat expansion in the C9orf72 gene leads to the accumulation of toxic RNAs that form stable G-quadruplexes. Indeed, it was the original observation that C9orf72 repeat RNA interacts with several paraspeckle proteins in a G-quadruplex-dependent manner that prompted the researchers to investigate the role of G-quadruplexes in paraspeckle formation.

To test their hypothesis, the authors began by studying an RNA-binding protein called NONO, which is recruited to the paraspeckles by NEAT1. They found that NEAT1 recruitment of NONO was increased under the conditions favoring the formation of G-quadruplexes. The binding of NONO to the G-quadruplexes was specific to that structure as identical RNAs not in the form of quadruplexes had a much lower affinity for the protein.

The researchers found that the NEAT1 lncRNA also contains numerous G-quadruplex motifs, many of which were clustered around the ends of the RNA. Binding experiments showed that NONO recognized the G-quadruplexes in NEAT1 both in vitro and in live cells, and that this recognition was crucial to their association. Importantly, when they examined the equivalent NEAT1 lncRNA in other species, they found that although the nucleic acid sequence didn’t seem to correspond, all of the NEAT1 homologues were enriched in G-quadruplexes.

Taken together, these results show the importance of G-quadruplexes in the formation of paraspeckles, and also provide another avenue by which the C9orf72 repeat expansion could interfere with normal cellular functioning.

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