Our cells turn genetic instructions into proteins with great precision. Critical for the translation of this information are transfer RNAs, or tRNAs, small molecules that read the genetic code three letters at a time and bring the matching building blocks of proteins, the amino acids, to the cell's protein-making machinery, the ribosome.
Every cell holds millions of tRNAs, but only some are busy at work in the ribosome. Until now, scientists have studied them by measuring all of the tRNAs in a cell at once, assuming this gives a fair picture of the ones actually doing the job.
In a new study in Nature Methods , researchers at the Centre for Genomic Regulation (CRG) in Barcelona describe a technique that looks only at the tRNAs on ribosomes busy making proteins. It shows that the tRNAs at work can differ from the cell's overall stock, particularly when cells are under stress, a difference that standard techniques miss.
"The field has largely assumed that sequencing all the tRNAs in a cell gives a good picture of the ones working in protein production. Our work shows that this is not necessarily the case, particularly under stress," said Dr Mie Monti, the study's first author.
When cells were starved of leucine, one of the building blocks of protein, the team saw the tRNAs piling up on the ribosome, when the expectation would be for tRNAs to disappear entirely.
The results suggest some changes inside cells may have gone unnoticed. Cancer cells, for example, are known to use tRNAs differently from healthy cells, but Dr Monti says this had only been studied by counting every tRNA in the cell.
Human cells also carry several versions of many tRNAs. Using viral infections as an example, Dr Monti says the new approach could show exactly which tRNA molecule a virus relies on. In principle, that could point to new ways of blocking an infection.
The biggest implication of the study is that it might accelerate the development of a new kind of medicine for genetic diseases.
About one in 10 of the genetic errors known to cause inherited disease are typos that tell the cell to stop making a protein too early. Specially designed tRNAs can read past the false stop sign and restore the full protein without changing a patient's DNA.
Experimental tRNA medicines have already restored missing proteins in cells and mice in studies of cystic fibrosis and Duchenne muscular dystrophy, and one was cleared this year for its first trial in people. Because the same typo can appear in many different genes, one tRNA medicine could in theory treat many unrelated conditions.
"A single tRNA medicine could, in principle, be used to treat multiple unrelated rare diseases. This would be transformative for rare disease patients, as these groups are otherwise too fragmented for conventional drug development," says ICREA Research Professor Eva Novoa, who led the study.
The method could also help those designing such medicines. "One thing is to design a tRNA that works in a test tube. Another is to know that it will actually go to the ribosome in sufficient amounts to be used by the cell," explains Dr Monti.
The authors of the study expect their new method, called tRIBO-seq, will become a routine tool for finding the most promising candidate medicines. The team co-developed it with the Italian company IMMAGINA Biotechnology.
The method works by fishing for ribosomes that are busy making proteins in a cell. The tRNAs inside are then read one by one, showing which are in use, which chemical tags they carry and whether any are damaged, all in a single experiment.
Conventional methods require about a week of growing cells and need specialist equipment. The CRG's method needs a single dish of cells and about a day and a half.
