tRNA-MaP: next-generation DNA sequencing for functional analysis of RNA-associated enzymes

The genetic information encoded in genomic DNA is transcribed into mRNA, which is then transferred during protein synthesis (tRNAs) to decode codons
on the mRNA.
tRNAs deliver amino acids to the ribosome and synthesize proteins
from amino acids on the ribosome based on the decoded genetic information.
Therefore, tRNA plays a key role
in the process of translating genetic information.
tRNAs contain many modified nucleosides that regulate the accuracy and efficiency
of protein synthesis.
The modified nucleosides in tRNA are synthesized
by tRNA modifying enzymes.
Thus, reveal the mechanism by which tRNA modifiers selectively recognize substrate tRNA from non-substrate RNA; When, where, and how much trna is modified by modifying enzymes is critical
to understanding the mechanisms of protein synthesis.
However, addressing these critical questions is challenging
due to the lack of a high-throughput technique to identify the characteristic properties of tRNA-modifying enzymes.

To overcome this problem, Yamagami and Hori applied next-generation DNA sequencing technology to functional analysis of tRNA modifiers and developed a new high-throughput analysis method "tRNA-map"
.
tRNA-map technology enables rapid screening of RNA pools consisting of more than 5,000 RNAs and identifies the substrate tRNA of the tRNA modifier enzyme of interest, which is relatively sensitive
compared to existing methods.

"With tRNA-MaP, combined with protein orthopedic analysis, we predicted a large number of naturally modified Geobacillus stearothermophilus
.
In addition, we analyzed the substrate recognition mechanism g.
stearothermophilustRNA ^m1A22 methyltransferase (TrmK), methylates adenosine at position 22 to 1-methyladenosine (^m1A22) in tRNA, using tRNA-map
.
Mutation analysis showed that TrmK selected a subset of trna as the substrate
.
Use 240 variants g .
stearothermophilus tRNA ^{low-enriched uranium} We found U8, A14, G15, G18, G19, U55, Purine57 and A58 important
for methylation of TrmK.
In addition, based on the recognition site in tRNA and the crystal structure of TrmK, the docking model
of TrmK and tRNA was constructed.
”

This study revealed that tRNA-maps can be used for analysis
of tRNA modifying enzymes.
Of note, since tRNA-maps can analyze any RNA molecule species, even DNA molecules, in any organism, tRNA-map can be used to analyze all nucleic acid-associated proteins
except tRNA modifiers.
Thus, tRNA-Map can accelerate the comprehensive understanding
of genetic information flow.