Conventional wisdom once held that each gene produced only a single protein. As we now know, the one gene-one protein hypothesis in molecular biology is a gross oversimplification. One gene can give rise to several ribonucleic acid (RNA) transcripts that have similar sequences and, occasionally, different functions. Known as RNA isoforms, these transcripts are the equivalent of molecular siblings.
It turns out that although RNA isoforms arise from the same gene, they are regulated in a variety of ways. For instance, they can be decayed at different speeds. What governs the differences in regulation, however, is still largely unknown, and a clue may lie within their three-dimensional structures.
“Traditionally, mapping RNA structures of each isoform is difficult because different isoforms from the same gene still look very similar at the sequence-level,” said study co-corresponding author Yue Wan, a Principal Investigator at A*STAR’s Genome Institute of Singapore (GIS).
Unlike current sequencing technologies which can only generate short sequences or reads,
a new method developed by Wan and her GIS colleague and co-corresponding author Niranjan Nagarajan, in collaboration with Meng How Tan from Nanyang Technological University, could revolutionize the study of RNA-based gene regulation.
Their technique, dubbed by the researchers as PORE-cupine, owes its unique name to nanopore sequencing. Because it can generate long reads, it is much easier to map out which RNA isoform the reads belong to.
“In nanopore sequencing, current flows through biological pores when an RNA molecule threads through,” explained Wan. “By detecting current changes through the pores, we are able to determine which base along the RNA molecule has been modified by chemical compounds, and hence whether it is double- or single-stranded.”
Testing PORE-cupine on human embryonic stem cells, the team showed that structural differences among the RNA isoforms could alter the amount of proteins made within cells. They also found that shared sequences in different RNA isoforms of the same gene can fold into different structures.
Aside from providing scientists with a new way of studying RNA-based gene regulation, PORE-cupine is also notable for its simplicity. After all, the method only involves two steps: modifying the RNA isoforms with structural probes and subjecting these isoforms to nanopore sequencing, with no amplification step required.
Moving forward, the team aims to further refine the technique’s machine learning algorithms. “We aim to test more structure-modifying compounds and additional types of machine learning strategies to figure out if there are other compounds or methods that can perform even better,” said Wan.