# tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators
## 3 Chemical Modifications: Guardians of tRNA Integrity and Function
### RNA Modification on tRNA
#### 3.1.1 5-Methylcytidine (m\({}^{5}\)C)  
m\({}^{5}\)C modifications are distributed on multiple positions of tRNA and modified by diverse modification enzymes, such as NOP2/Sun RNA methyltransferase 2/3/4/6 (NSUN2/3/4/6) [44; 45; 46] and tRNA (cytosine(38)-C(5))-methyltransferase transferase TRDMT1/DNMT2 [47]. Additionally, alpha-ketoglutate-dependent dioxygenase homolog 1 (ALKBH1) was reported to further oxidize  
Figure 2: RNA modification on tRNAs and biogenesis of tsRNAs. (**a**) Possible tRNA modification types, sites, and corresponding enzymes. (**b**) 5\({}^{\prime}\)tsRNAs generate from D loop nuclease or anticodon loop nuclease; 3\({}^{\prime}\)tsRNAs generate from TYC loop or anticodon loop nuclease; inner\({}^{\prime}\)tsRNAs generate from D loop and TYC loop nuclease.  
m\({}^{5}\)C to 5-formylcytidine (f\({}^{5}\)C), while Ten-eleven translocation methylcytosine dioxygenase 2 (TET2) oxidizes m\({}^{5}\)C to 5-hydroxymethylcytidine (hm\({}^{5}\)C) [48; 49]. Evidence has shown that m\({}^{5}\)C can protect tRNAs from fragmentation by nucleases. For example, DNMT2 could methylate C38 to m\({}^{5}\)C38 on tRNA\({}^{Asp}\), tRNAVal, tRNAGlu, tRNAGln, and tRNAGly, which further stabilized these tRNAs from cleavage at the anticodon site [47; 50; 51]. The genetic deletion of _Dnmt2_ in mouse testis and sperm facilitates the biogenesis of tsRNAs [52]. In addition to DNMT2, the NSUN family shows a broader methylation range of tRNA m\({}^{5}\)C, of which NSUN2 is responsible for the generation of m\({}^{5}\)C at positions 48, 49, and 50 of tRNA [53], while NSUN6 is responsible for the methylation of C72 of tRNA\({}^{Thr}\) and tRNA\({}^{Cys}\)[46; 54]. NSUN3 is responsible for the initiation generation of m\({}^{5}\)C at the wobble position of mt-tRNA\({}^{Met}\)[55], which is further oxidized by ALKBH1 to generate 5 fC [56]. The mt-tRNA\({}^{Met}\) with 5 fC34 is capable of reading AUA codons as methionine in mitochondria. The existence of NSUN3 and ALKBH1 is pivotal for cells to enhance the translation rate via 5 fC34 mt-tRNA\({}^{Met}\) to maintain the function of mitochondria. Moreover, the aberrance of other NSUN family proteins can trigger a variety of downstream dysregulations due to the absence of m\({}^{5}\)C modification on tRNAs. Notably, the deletion of _NSun2_ in testis destabilizes tRNAs and reduces the levels of several tRNAs, which further impairs spermatogenesis by modulating the translation of RNA processing proteins [57]. In addition, the deficiency of NSUN2 in neurons significantly decreases the abundance of tRNA\({}^{Gly}\) and subsequently suppresses the translation of Gly-rich proteins, which ultimately leads to impaired synaptic signaling [51]. A summary of m\({}^{5}\)C and other tRNA modifications related to pathologies is shown in Table 1.  
In light of the evidence for the demethylation of tRNA m\({}^{5}\)C, Tet family proteins were identified as being responsible for the oxidization of m\({}^{5}\)C on tRNAs. Particularly, TET2 showed a strong affinity with tRNA m\({}^{5}\)C, especially tRNA\({}^{Gly}\), and could oxidize tRNA m\({}^{5}\)C to hm\({}^{5}\)C both in vivo and in vitro. The deletion of _Tet2_ in mouse embryonic stem cells (mESCs) significantly reduced the abundance of tRNA hm\({}^{5}\)C, while overexpression of the catalytic domain of TET2 increased the abundance of tRNA hm\({}^{5}\)C, with an obvious decrease in tRNA m\({}^{5}\)C [48], suggesting a direct functional role of TET2 on tRNA.