# tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators
## 8 Conclusions and Perspectives  
The diversity and heterogeneity of tRNA have led to a significant advancement in our understanding of the tRNA world. In fact, the organisms have evolved multiple redundancies of tRNA genes to ensure the reliable functioning of tRNAs. The number of tRNA genes varies among different species, with mice, humans, and other primates having between 400 and 700 tRNA genes, while zebrafish have approximately 10,000 [194]. In addition to the considerable diversity of tRNA gene copy numbers, the cellular tRNA pool is also intricately regulated. On the transcription level, the gene location of specific tRNAs, chromatin accessibility, activity of the Pol III complex, and selective binding of transcription factors play pivotal roles in regulating the transcription initiation of tRNA genes [17; 36; 195]. After transcription, the maturation of tRNAs is also tightly controlled by a complex network that consists of RNAases, CCA tailoring processes, RNA modification enzymes, and aminoacyl-tRNA synthetases [196]. These regulatory processes exert influences over the composition of the tRNA isoacceptors and isodecoders within the cell, forming cell-specific tRNA pools [35; 161]. For instance, external alterations could influence tRNA total abundance, while specific tRNA isoacceptor abundance could regulate translation rates to meet tissue-specific translation needs. Additionally, tRNA modifications also play an important role in the formation and maintenance of the tRNA cloverleaf-like secondary structure and L-shaped tertiary structure, while some tRNA modifications are subjected to intricate regulation during translation, which governs the fidelity and efficiency of codon-anticodon recognition, conveying information beyond the tRNA sequence [197; 198; 199]. Furthermore, the non-classical functions of tRNAs encompass the regulation of transposon activity, modulation of tRNA gene expression, and participation in cellular immune regulation and viral replication. These observed biological functions illustrate the complex regulatory roles of tRNAs. It is noteworthy that tRNAs are also capable of generating tRNAs by fragmentation in response to cellular requirements [26]. This process allows tRNAs to participate in biological regulation in the form of a new regulatory non-coding small RNA. As expected, tRNAs exhibit multifaceted regulatory effects at the transcriptional, post-transcriptional, and translational levels through diverse mechanisms [200]. This regulatory strategy is highly sophisticated, whereby intact tRNAs engage in regulatory functions, both classical and non-classical, and, when necessary, generate new sncRNAs to meet the regulatory demands of the cell, thus enabling an efficient and multifaceted mode of regulation. In recent years, the establishment of new high-throughput technologies for detecting tRNA and tsRNA profiles has provided us with a glimpse into the tRNA and tsRNA world during various physiological and pathological processes, unveiling the potential application of tRNA and tsRNA as diagnostic biomarkers and therapeutic strategies for various diseases [182; 201; 202]. Studies have already demonstrated that the manipulation of tRNAs can be used to treat genetic diseases [193; 203]. Similarly, as a novel class of small RNAs, tsRNAs show great potential for application in disease diagnosis. However, several methodological challenges remain on the path to practical applications. Firstly, how can we achieve highly accurate high-throughput detection of every type of modification on tRNA? Due to the complexity and diversity of tRNA, current technologies sometimes struggle to accurately distinguish between modification types or sites. In the future, analyses of tRNA heterogeneity and tsRNA functions, much like mRNA research, are expected to become a routine part of multi-omics studies. A deeper and more detailed understanding of the tRNA landscape may allow us to gain a \"[PERSON]'s eye view,\" allowing for the early prediction of cell fate or disease progression.