Ribonucleic acid (RNA) is a key molecule that plays important roles in regulating gene expression in cells. Although scientists have extensively studied how mini cellular machines such as proteins and chromatin fold and interact with each other over the past decades, less is known about how RNA comes together to interact with itself or with other RNA molecules, or the impact on gene regulation of RNA.
Now, researchers in Singapore have developed a new high throughput method that identifies how ribonucleic acid molecules come together inside cells. Named SPLASH (for Sequencing of Psoralen Linked And Selected Hybrids), this new technique was used to describe the RNA network in human and yeast cells, its dynamics, and how the structural organisation impacts translation and decay processes in the cell.
In the past, to investigate connectivity information for linking nucleotides that are physically pairing, various RNA crosslinkers, such as methylene blue, UV, and psoralen, have been used to connect faraway interacting regions of RNAs to each other. Feedback from these methods has typically been slow and laborious, however.
Other recent strategies such as the proximity ligation-based approach RPL, or high-throughput sequencing methods such as CLASH, Hi-CLIP, and RAP, also have their limitations. SPLASH employs psoralen crosslinking to identify RNA interactions in vivo, nonselectively and genome-wide.
Key to Cellular Function
The development was jointly led by Drs Wan Yue and Niranjan Nagarajan from A*STAR’s Genome Institute of Singapore.
Co-lead author Dr Wan, said:
“The cell is a complex machine; we need to understand the configuration of all its components to be able to engineer and/or repair it. RNA shapes and RNA interaction networks are key to cellular function. Depending on cellular needs, these dynamic interaction networks can be remodelled. Most importantly, targeting these networks could be a means to inhibit infectious organisms.”
SPLASH will enable the team to study the transcriptomes of infectious organisms, including pathogenic bacteria, dengue and Zika viruses, to understand the way RNA shapes and networks in these genomes enable the infection of human cells by pathogens. It is hoped that these efforts in understanding microbial pathogenicity will contribute to new anti-microbials, anti-virals or vaccines against these pathogens.
As the authors summarize in their paper:
SPLASH expands our understanding of the structural organization of eukaryotic transcriptomes, and helps to define the principles of how RNAs interact with themselves and with other RNAs in gene regulation and ribosome biogenesis.
We anticipate that future studies using SPLASH and its variants will continue to shed light on the complexity and dynamics of RNA interactions in cellular systems across diverse organisms.
Jong Ghut Ashley Aw, et al.
In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation
Molecular Cell (2016). DOI: 10.1016/j.molcel.2016.04.028
Image: MRC Lab of Molecular Biology, Wellcome Images