In biology’s standard tale of two acids — deoxyribonucleic (DNA) and ribonucleic (RNA) — RNA is merely the messenger.
Its better-known cousin DNA plays the lead role, issuing genetic instructions for the construction of complex molecules called proteins that do the cells’ work. Recent research, however, shows that specialized proteins that bind to RNA also regulate the messages RNA delivers.
Mutations in the nearly 2,100 genes that encode RNA-associated proteins occur in cancer, neurodegeneration, developmental disorders and other diseases. But it’s been difficult to pin down what each of those proteins is doing within the cell.
Researchers at Fred Hutch Cancer Center invented a new screening method called ReLiC — described recently in the journal Nature Methods — that solves that problem using CRISPR-Cas9, the Nobel-prize winning gene-editing technology.
Instead of knocking out the genes that encode RNA-associated proteins one at a time, ReLiC tests them all at once on a batch of cells, each cell edited so that it’s missing only one key gene.
ReLiC measures how those gene deletions affect various RNA processes that are traditionally difficult to measure at scale.
“This approach has opened up a new way for us to systematically interrogate RNA regulation,” said Arvind (Rasi) Subramaniam, PhD, a researcher in the Basic Sciences Division who joined Fred Hutch 10 years ago and studies how cells make proteins from RNA.
Many things influence RNA, from transcription to translation to degradation
Messenger RNA is a go-between molecule that copies DNA codes for building proteins — a process called transcription — and then delivers the transcripts to the cell’s many protein factories, which are called ribosomes.
In the ribosomes, the RNA sequences are translated into amino acid sequences, which are then used to make proteins.
RNA-associated proteins influence RNA’s function from the moment a messenger RNA molecule is created during transcription to when it is translated in the ribosome all the way to its eventual degradation when it’s no longer needed.
“But which of these proteins in the cell are actually doing the job, and what exactly are they doing?” Subramaniam asked.
One way to find out is to delete a gene that codes for an RNA-associated protein and see what changes.
“You can do it for a few genes but doing it for more than that gets really unwieldy,” he said.
What the field needed was a screening technique flexible enough to identify the functions of thousands of RNA-associated proteins at once without breaking the bank.
As part of his doctoral thesis research, Patrick J. Nugent, PhD, a former student in the Subramaniam Lab, took on that challenge and helped develop the method they call ReLiC (the capital letters stand for RNA-Linked-CRISPR).
Using CRISPR to reveal hard-to-reach RNA processes
The ReLiC method relies on a molecular tool called site-specific integration to deliver a customized set of DNA sequences called a library to a specific location in the human genome.
“The site-specific integration method helps us keep everything the same except the gene that we are deleting,” Subramaniam said. “That’s what makes it powerful. We can knock out about 2,000 human genes in a single Petri dish.”