# tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators
## 3 Chemical Modifications: Guardians of tRNA Integrity and Function
### Rebirth of tsRNA from tRNA  
The generation of tsRNAs in cells primarily depends on the recognition and cleavage of tRNAs by endogenous nucleases (Figure 2b). These specific nucleases are capable of recognizing tRNAs based on their modification status and cleaving them at specific locations, producing tsRNA fragments with diverse lengths and sequences. Researchers found that most tsRNAs originate from mature tRNAs, but a small portion of tsRNAs are derived from precursor tRNAs. During the maturation process of precursor tRNAs (Pre-tRNAs), ribonuclease P (RNase P) can remove the 5\({}^{\prime}\) end sequences of Pre-tRNAs to generate 5\({}^{\prime}\)-Leader-tsRNAs, while ribonuclease Z (RNase Z) and ElaC homolog protein 2 (ELAC2) can cleave the 3\({}^{\prime}\) ends of Pre-tRNAs to produce 3\({}^{\prime}\)-Trailer-tsRNAs [128]. In the mammalian nuclease A family, ANG can cleave near the anticodon loop of mature tRNAs under stress conditions, producing 5\({}^{\prime}\)tsRNAs and 3\({}^{\prime}\)tsRNAs (also known as 5\({}^{\prime}\)tRNA halves and 3\({}^{\prime}\)tRNA halves) with lengths around 30-40 nucleotides. Ribonuclease Dicer can cleave near the D loop and TYC loop to produce shorter 5\({}^{\prime}\)tsRNAs and 3\({}^{\prime}\)tsRNAs with lengths of around 18-25 nucleotides [129]. Additionally, RNase P can also target specific sequences (such as those GC-rich base pairs) in the stem of the tRNA anticodon loop for cleavage [130]. Studies have revealed a universal expression pattern of tsRNA in various tissues and cell types among animals and plants. Interestingly, the profiles of tsRNA show significant expression heterogeneity among different tissues, body fluids, and cancer samples. For instance, in terms of the total amount, tsRNAs are highly enriched in serum and mature sperm, in which serum tsRNAs are dominantly encapsulated by the serum protein complex and sensitive to body active infection and inflammation, while sperm tsRNA exhibits enrichment in the sperm head [131; 132] and sensitively responds to environmental exposures, nutrition alteration, mental trauma, and aging. Moreover, besides serum and sperm, growing evidence shows considerable tsRNA abundance in various cancers with distinct signatures, suggesting a promising application direction of tsRNAs as diagnostic biomarkers \begin{table}
\begin{tabular}{c c c} \hline \hline
**tRNA Modification** & **Associated Pathology** & **References** \\ \hline \multirow{3}{*}{m\({}^{5}\)C} & Male germ cell differentiation & [57] \\  & Tumor metastasis & [122] \\  & Parametally acquired metabolic disorders & [52] \\ \hline \multirow{3}{*}{m\({}^{1}\)A} & T cell proliferation post-antigen stimulation & [123] \\  & Mitochondrial disorders: MERRF (myoclonus & [124] \\  & epilepsy, ragged-red fibers) & [125] \\ \hline \multirow{3}{*}{m\({}^{1}\)G} & Ehrlich ascites tumor; neuroblastoma & \\  & Hereditary spastic parapresis & [126] \\  & Neuropathy syndromes & [78] \\  & Combined oxidative phosphorylation deficiency & [79] \\  & 26 (COXPD26) & [80] \\  & Idiopathic non-cirrhotic portal hypertension; & [81] \\  & hepatopulmonary syndrome & \\ \hline \multirow{3}{*}{m\({}^{7}\)G} & Acute myeloid leukemia & [86; 92] \\  & Bladder cancer & [93] \\  & Hepatocellular carcinoma & [94] \\  & Prostate cancer & [95] \\  & Stem cell fate determination & [16] \\  & Aging & [97] \\ \hline \multirow{3}{*}{m\({}^{3}\)C} & Human developmental delay and epileptic & [103] \\  & encephalopathy & [127] \\ \hline \multirow{3}{*}{Q} & Sex-dependent learning and memory formation & [121] \\  & in the hippocampus & [121] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: tRNA modifications and their associated pathologies. ers of cancers.