# tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators
## 3 Chemical Modifications: Guardians of tRNA Integrity and Function
### RNA Modification on tRNA
#### 3.1.5 N3-Methylcytidine (m\({}^{3}\)C)  
On human tRNA, m\({}^{3}\)C usually occurs at various positions such as C32, C20, and C47:3 on cytosol tRNA (cyto-tRNA), while only one position of m\({}^{3}\)C32 was identified on mt-tRNA [98]. The methylation of m\({}^{3}\)C on human cyto-tRNA\({}^{\rm Thr}\) and cyto-tRNA\({}^{\rm Arg}\) is catalyzed by METTL2A/METTL2B, while METTL6 catalyzes m\({}^{3}\)C32 on human cyto-tRNA\({}^{\rm Ser}\)[99]. However, the methyltransferases that are responsible for m\({}^{3}\)C20 of human cyto-tRNA\({}^{\rm Ser}\) and cyto-tRNA\({}^{\rm Leu}\) and m\({}^{3}\)C47:3 of human cyto-tRNA\({}^{\rm Met}\) are still unknown.  
Importantly, the methylation of m\({}^{3}\)C occurs on the Watson-Crick interface of cytosine and endows a positive charge on the nucleobase. This chemical character of m\({}^{3}\)C significantly alters its topological structure and base-paring capacity, which blocks the reverse transcription by high-fidelity reverse transcriptase or induces mismatch substitutions by low-fidelity reverse transcriptase during the cDNA synthesis process [100]. Moreover, the methylation of m\({}^{3}\)C32 by its modifiers requires a sequence context for the recognition of appropriate target tRNAs, such as human METTL2A/METTL2B methylate m\({}^{3}\)C32 of cyto-tRNA\({}^{\rm Thr}\) and cyto-tRNA\({}^{\rm Arg}\) in the presence of G35 and U36, respectively. Moreover, the modification state of A37 also plays a necessary function in the formation of m\({}^{3}\)C32 [101], suggesting a mutual regulation and crosstalk network for cellular tRNA modification control.  
Functionally, increasing evidence shows the important role of tRNA m\({}^{3}\)C32 modification in regulating cellular translation rate and mitochondrial protein synthesis [98]. The deletion of METTL2A/METTL2B and METTL6 decreased the level of m\({}^{3}\)C32 modification on tRNA\({}^{\rm Ser(GCT)}\) and altered the translation of serine codon-biased mRNA [102]. Knockdown of a cofactor of METTL2, DALR anticodon binding domain containing 3 (DALRD3), leads to nearly complete loss of m\({}^{3}\)C32 on cyto-tRNA\({}^{\rm Arg}\), which might be associated with human developmental delay and epileptic encephalopathy [103].  
Recently, METTL8 was found to methylate C32 to m\({}^{3}\)C32 for mt-tRNAThr and mt-tRNASer[104; 105; 106] and be involved in regulating the maintenance of embryonic cortical neural stem cells in mice [107]. The modification of m\({}^{3}\)C32 promotes mitochondria tRNA folding and maintains tRNA structure stability, thereby enhancing the translation efficiency of mitochondrial proteins. Interestingly, the biological role of METTL8 is not restricted to mt-tRNA m\({}^{3}\)C32 modification; the generation of various METTL8 isoforms by alternative splicing diversifies the biological function of METTL8. As reported, some METTL8 isoforms could enter into mitochondria and serve as tRNA m\({}^{3}\)C32 modifiers, while some isoforms are retained in the nucleolus and function as a regulator in R loop formation [108].