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Transfer RNAs (tRNA) are essential components of the translation process and are frequently modified to perform various functions1,2. Modified tRNAs are very common, as the average tRNA contains 13 modifications3. Additionally, studies have shown that these modifications may have a role in different diseases4,5.
Next-generation sequencing and mass spectrometry are typically the preferred methods for measuring tRNA, but they are limited in their ability to accurately quantify their abundance and chemical modifications, hindering important tRNA research.
A new study in Nature Biotechnology overcomes the previous limitations of studying tRNAs by using a nanopore-based method to sequence native tRNA populations. The new method, called Nano-tRNAseq, allows users to measure the abundance and tRNA modifications in one step without the need for amplification or other preparation steps that can introduce biases into the process.
The work capitalizes on Oxford Nanopore Technologies’ (ONT) direct RNA sequencing platform that can determine individual bases and their modifications.
Nano-tRNAseq begins with a library preparation incorporating a double ligation of RNA adapters at both the 5′ and 3′ ends of the tRNA molecules, benefiting from the 3′ CCA overhang normally present in the mature tRNAs. This improved library preparation led to an increased proportion of basecalled and mapped tRNA molecules. However, the proprietary ONT software, MinKNOW, often registers tRNAs as adapter sequences and causes biases in the estimated tRNA abundances. To counteract this, the researchers developed a computational framework that captures roughly 10× more tRNA reads and recapitulates tRNA abundances.
To test their new method, they used Nano-tRNAseq to measure tRNA abundances and modifications from yeast cells exposed to different conditions. The results showed that Nano-tRNAseq has many advantages over other common methods. Although, the researchers have noted that the estimations of tRNA abundances generated are limited to those tRNAs known and used as references in the mapping step.
“For the first time, we can study both tRNA abundance and tRNA modification profiles simultaneously,” said Morghan Lucas, the first author of the study and Ph.D. candidate at the Centre for Genomic Regulation. “As a bonus, the method is rapid, cost-effective, high-throughput, and has single-molecule resolution. Previously, we relied on two separate methods that, together, are less informative, and it would take weeks and cost thousands of euros to obtain results. Nano-tRNAseq is a fraction of the cost, and we can have results within a couple of days, and in the near future, within a few hours.”
The quick and accurate analysis offered by Nano-tRNAseq will allow researchers to greatly enhance their tRNA studies and potentially utilize it for clinical work in the future.
“tRNA molecules can be cleaved into small but stable RNA fragments which circulate in blood plasma,” said Dr. Eva Novoa, researcher at the Centre for Genomic Regulation and the senior author of the study. “These molecules are typically altered in cancer patients, and are hugely information-rich for diagnostic and prognostic purposes. Nano-tRNAseq is a proof-of-concept technology that paves the way for the development of a simple, cost-effective and highly-precise method that can quantify these molecules in a non-invasive manner. Our aim is to further develop this technology and combine it with artificial intelligence tools to determine the malignancy of a biological sample in less than 3 hours, and at a cost of no more than 50 euros per sample.”
References:
Next-generation sequencing and mass spectrometry are typically the preferred methods for measuring tRNA, but they are limited in their ability to accurately quantify their abundance and chemical modifications, hindering important tRNA research.
A new study in Nature Biotechnology overcomes the previous limitations of studying tRNAs by using a nanopore-based method to sequence native tRNA populations. The new method, called Nano-tRNAseq, allows users to measure the abundance and tRNA modifications in one step without the need for amplification or other preparation steps that can introduce biases into the process.
The work capitalizes on Oxford Nanopore Technologies’ (ONT) direct RNA sequencing platform that can determine individual bases and their modifications.
Nano-tRNAseq begins with a library preparation incorporating a double ligation of RNA adapters at both the 5′ and 3′ ends of the tRNA molecules, benefiting from the 3′ CCA overhang normally present in the mature tRNAs. This improved library preparation led to an increased proportion of basecalled and mapped tRNA molecules. However, the proprietary ONT software, MinKNOW, often registers tRNAs as adapter sequences and causes biases in the estimated tRNA abundances. To counteract this, the researchers developed a computational framework that captures roughly 10× more tRNA reads and recapitulates tRNA abundances.
To test their new method, they used Nano-tRNAseq to measure tRNA abundances and modifications from yeast cells exposed to different conditions. The results showed that Nano-tRNAseq has many advantages over other common methods. Although, the researchers have noted that the estimations of tRNA abundances generated are limited to those tRNAs known and used as references in the mapping step.
“For the first time, we can study both tRNA abundance and tRNA modification profiles simultaneously,” said Morghan Lucas, the first author of the study and Ph.D. candidate at the Centre for Genomic Regulation. “As a bonus, the method is rapid, cost-effective, high-throughput, and has single-molecule resolution. Previously, we relied on two separate methods that, together, are less informative, and it would take weeks and cost thousands of euros to obtain results. Nano-tRNAseq is a fraction of the cost, and we can have results within a couple of days, and in the near future, within a few hours.”
The quick and accurate analysis offered by Nano-tRNAseq will allow researchers to greatly enhance their tRNA studies and potentially utilize it for clinical work in the future.
“tRNA molecules can be cleaved into small but stable RNA fragments which circulate in blood plasma,” said Dr. Eva Novoa, researcher at the Centre for Genomic Regulation and the senior author of the study. “These molecules are typically altered in cancer patients, and are hugely information-rich for diagnostic and prognostic purposes. Nano-tRNAseq is a proof-of-concept technology that paves the way for the development of a simple, cost-effective and highly-precise method that can quantify these molecules in a non-invasive manner. Our aim is to further develop this technology and combine it with artificial intelligence tools to determine the malignancy of a biological sample in less than 3 hours, and at a cost of no more than 50 euros per sample.”
References:
- Novoa, Eva Maria, and Lluís Ribas de Pouplana. "Speeding with control: codon usage, tRNAs, and ribosomes." Trends in Genetics 28.11 (2012): 574-581.
- Motorin, Yuri, and Mark Helm. "tRNA stabilization by modified nucleotides." Biochemistry 49.24 (2010): 4934-4944.
- Pan, Tao. "Modifications and functional genomics of human transfer RNA." Cell research 28.4 (2018): 395-404.
- Jonkhout, Nicky, et al. "The RNA modification landscape in human disease." Rna 23.12 (2017): 1754-1769.
- Torres, Adrian Gabriel, Eduard Batlle, and Lluis Ribas de Pouplana. "Role of tRNA modifications in human diseases." Trends in molecular medicine 20.6 (2014): 306-314.