# tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators
## 3 Chemical Modifications: Guardians of tRNA Integrity and Function
### RNA Modification on tRNA
#### 3.1.2 N1-Methyladenosine (m\({}^{1}\)A)  
The m\({}^{1}\)A modification on tRNAs occurs at positions m\({}^{1}\)A9, m\({}^{1}\)A14, m\({}^{1}\)A22, m\({}^{1}\)A57, and m\({}^{1}\)A58 of different tRNAs across many life forms from bacteria to mammals [58; 59]. Among them, m\({}^{1}\)A58 is the most universal methylation position that occurs on tRNAs across all three kingdoms of life, while the other positions of m\({}^{1}\)A are relatively rare. Due to the chemical characteristic of m\({}^{1}\)A, m\({}^{1}\)A could block the Watson-Crick base pairing and play an important role in maintaining tRNA structural stability and folding correction. A lack of m\({}^{1}\)A9 and/or m\({}^{1}\)A58 reduces the thermostability of tRNA L-shaped tertiarily structures and induces the cleavage of tRNAs to generate tsRNAs. In addition, m\({}^{1}\)A is a reversible nucleotide chemical modification, which can be \"written\" and \"erased\" by its corresponding enzymes. The tRNA Methyltransferase6/61 (Trm6/Trm61) serves as the writer of m\({}^{1}\)A58 modifications [60], while ALKBH1/2/3/7 and fat mass and obesity-associated proteins (FTO) function as the erasers of m\({}^{1}\)A. The deletion of m\({}^{1}\)A writer Trm61A in thymocyte induces a global decrease in tRNA m\({}^{1}\)A58 modification abundance and impairs the translation efficiency of Myc mRNA, preventing the proliferation of T cells following antigen stimulation [61; 62]. The TRMT6/61A complex also shows dimethylation capacity that methylates tRNA A58 into m\({}^{1,6}\)A58.  
ALKBH1 and ALKBH2/3 were first reported to function as DNA repair proteins; however, increasing evidence has shown that they can serve as erasers of tRNA m\({}^{1}\)A modification. For instance, ALKBH1 was reported to demethylate tRNA m\({}^{1}\)A inside cells and tune translation initiation and elongation by modulating the cellular abundance of tRNA\({}^{Met}\) and other m\({}^{1}\)A-modified tRNAs that enter the translation machine [63]. ALKBH2/3 were reported to not only show significant m\({}^{1}\)A demethylation capacity on various tRNAs but also on mRNA and CAG repeat RNA. Moreover, ALKBH2/3 can also demethylate m\({}^{3}\)Con tRNA, mRNA, and CAG repeat RNA [64; 65; 66; 67]. High levels of ALKBH3 in cancer cells decrease the abundance of m\({}^{1}\)A and m\({}^{3}\)C on tRNAs and promote cancer progression by facilitating the biogenesis of tsRNAs [68]. ALKBH7 was recently found to be capable of demethylating m\({}^{1}\)A and m\({}^{2}_{2}\)G of several pre-tRNAs in mitochondria and functioning as a regulator in mitochondria polydistronic RNA processing. Knockout of ALKBH7 impairs mitochondrial protein translation and suppresses mitochondrial activity by reducing the abundance of steady-state mitochondria-encoded tRNA within m\({}^{1}\)A and m\({}^{2}_{2}\)G modifications [69].  
Another m\({}^{1}\)A demethylase, FTO, was first discovered as an m\({}^{6}\)A eraser [70] and later confirmed to be able to demethylate m\({}^{1}\)A in tRNAs. Cross-Linking and Immunoprecipitation High Throughput Sequencing (CLIP-Seq) showed a strong binding affinity between FTO and tRNAs. Knockdown of FTO significantly increased the abundance of cellular m\({}^{1}\)A in tRNAs, while the overexpression of FTO led to a decrease in tRNA m\({}^{1}\)A levels [71]. Interestingly, the researchers also investigated the recognition strategy of FTO to distinguish different RNA species as demethylation substrates, especially for tRNAs. The results showed that FTO preferentially binds to stem-loop RNAs and shares a similar overall structure with NSUN6, which could bind to tRNA to methylate m\({}^{5}\)C72, thus promoting the demethylation efficiency of FTO to tRNA m\({}^{1}\)A58 [72].