How human transfer RNAs are made

Researchers led by Danny Nedialkova at the MPI of Biochemistry discover the mechanisms regulating tRNA expression in human cells, enabling the targeted development of tRNA-based drugs.

The human body consists of thousands of different cell types that each contain unique sets of proteins. The synthesis of these proteins relies on transfer RNAs (tRNAs), which deliver amino acids to ribosomes, the site of protein production. Errors in this process can lead to non-functional proteins and to human neurological diseases. Researchers led by Danny Nedialkova at the Max Planck Institute (MPI) of Biochemistry now discovered the mechanisms responsible for how different cell types maintain their tRNA composition during development. Their findings were published in the journal Nature Cell Biology.

To produce the proteins that are crucial for the well-being of our cells, the information stored in our DNA must be correctly decoded and translated. To do so, our DNA must be first transcribed into the so-called messenger RNA (mRNA). Although every cell in our body contains the same genetic information, different parts of this information are used in different cell types. Regulating which parts are used ensures that only the really necessary proteins are produced throughout the life of a cell. The mRNA blueprints are then read by ribosomes and assembled into proteins with the help of tRNAs, which are short noncoding RNA molecules with a cloverleaf-like structure. Each tRNA carries one of the twenty amino acids that serve as building blocks and binds to a codon specifying this amino acid in messenger RNA.

There are 61 codons for all twenty amino acids, but the number of tRNA molecules that decode them can differ substantially among organisms and cell types. Therefore, it has been challenging to determine how exactly the levels of individual tRNAs are regulated in human cells, as there are more than 600 genes in the human genome that code for tRNAs, many of which are found in several locations.

In addition, tRNA expression in mammals is highly selective and dynamically regulated, but it is not yet known exactly how this is achieved. Defining the mechanisms that control tRNA abundance is crucial for understanding how tRNA defects lead to human neurological diseases.

Measuring the (tRNA) world in human cells

The stable structure and abundant chemical modifications in tRNAs make it extremely challenging to measure their levels in cells using conventional approaches. Danny Nedialkova’s team recently overcame this technological barrier by developing a method called mim-tRNAseq, which quantifies tRNAs with unprecedented accuracy and resolution.  As a result, the team found that the gene copy number was not a very good predictor of tRNA abundance. This in turn raises the question of how human tRNA repertoires are controlled, and how much they vary among different cell types.

Not all human tRNA genes are equal

 “There has been this theory in the field that tRNA pools are tailored to the unique proteome of each cell type”, says Danny Nedialkova, Max Planck Research Group Leader at the MPI of Biochemistry and professor at the Department of Bioscience at the Technical University of Munich. “Because heart cells synthesize a lot of sarcomere proteins, maybe they need higher levels of specific tRNAs that are not so necessary in brain cells, for example. A bit like in a kitchen, where you would need more salmon in a sushi restaurant than in a bakery”, says Nedialkova further. 

To test this theory, co-first author Lexi Gao devised a workflow to differentiate human induced pluripotent stem cells into neuronal and heart cells in the lab. This enabled the researchers to study tRNA regulation in physiologically relevant cell models. “We were surprised to find that despite substantial remodeling of tRNA repertoires during differentiation, the abundance of tRNAs matching each mRNA codon was nearly the same across cell types and the usage of the different mRNA codons was also stable”, says Lexi Gao.

Together with co-first author Andrew Behrens, Gao went on to show that individual tRNA genes have very unequal expression. “Only about a third of human tRNA genes were expressed in all cell types. These genes – which we called ‘housekeeping’ – have distinct sequences that favor the recruitment of RNA polymerase III for transcription. Housekeeping tRNA genes also gave rise to the most abundant tRNAs in most cell types”, explains Behrens.

The stability of tRNA pools matching different codons across cell types could minimize the potential for ribosome errors during development. “A generic restaurant kitchen may have its advanatges after all, it’s no use having a fridge full of salmon when you need a chocolate cake”, says Nedialkova. ​​​​​​

From predictions to therapeutics

The rules governing tRNA expression identified by Nedialkova’s team have important implications for the emerging field of tRNA medicine. Engineering suppressor tRNAs that can bypass premature termination codons in mRNA could provide a treatment for thousands of human genetic diseases.

Until now, suppressor tRNA design has mostly focused on identifying molecules that can be efficiently charged with amino acids in cells, and that do not induce the readthrough of natural termination codons. “Our data show that the efficacy of suppressor tRNA therapeutics will also strongly depend on the choice of sequence elements that regulate RNA Polymerase III transcription”, concludes Danny Nedialkova.

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Dictionary of the Research Group Mechanisms of Protein Biogenesis:

mRNA: messenger RNA molecules that delivers the blueprint of proteins to the ribosome.

Ribosome: The protein-making machinery in cells, which translates the mRNA sequence blueprint into the corresponding amino acids to assemble a new protein.

tRNA: transfer RNA, short molecules that deliver specific amino acids to ribosomes during translation of messenger RNA into proteins

mim-tRNAseq: a method for accurately measuring the amount of each tRNA in cells developed by Danny Nedialkova's team. More information can be found here.

Human induced pluripotent stem cells: self-renewing cells derived by reprogramming human skin or blood cells to restore their capacity to differentiate into nearly any type of cell in the body.

Polymerase: an enzyme that assembles DNA or RNA molecules from their basic building blocks in cells.