# tRNA and tsRNA: From Heterogeneity to Multifaceted Regulators
## 4 Revisit the Regulatory Mechanisms of tRNA and tsRNA
### tsRNA Regulation: Structure Dictates Function
#### 4.2.2 tsRNA Secondary Structure  
In addition to a sequence-dependent regulatory mechanism, tsRNAs can also function independently of the sequence complementary principle but through its unique secondary or three-dimensional structures [155]. The spatial structure of some tsRNAs may present challenges during the small RNA sequencing process, potentially leading to the under-discovery of genuinely existing tsRNAs. Moreover, due to the highly modified characteristics of tsRNA, the investigation of tsRNA by using synthesized unmodified tsRNA mimics could not fully elucidate their sequence-independent functions, thereby impeding our understanding of the regulatory mechanisms of tsRNAs. However, growing evidence suggests that many tsRNAs indeed possess diverse secondary structures based on their sequence composition or various RNA modifications, and the regulatory function triggered by spatial interactions with other molecules should not be underestimated. On many occasions, the spatial conformation and chemical RNA modification signatures of tsRNAs contribute to the recognition and binding affinity by RNA binding proteins. For example, the ribonucleoprotein (RNP) of _Tetrahymena_ consists of spatially structured tsRNAs that bind to AGO/PIWI in an aptamer-like manner to promote the processing of nuclear rRNA [156; 157]. In mammalian cells, 5'tsRNAs containing a terminal oligo-G motif (TOG), such as 5'tsRNAAla and 5'tsRNACys (-30 nt), can fold into intermolecular RNA G-quadruplex (RNA G4) structures. The tsRNAs with RNA G4 structures can either competitively bind to the translation initiation complex eIF4G/eIF4E or bind to Y-Box Binding Protein 1 (YBX1) to prompt mRNA packaging into stress granules, thereby hindering translation initiation and resulting in global translation repression. Moreover, 5\({}^{\prime}\)tsRNA\({}^{\text{Ala/Cys}}\) possesses a \(\Psi\) modification at the position of \(\Psi\)8, which can also displace m\({}^{7}\)G-capped mRNA from polyadenylate binding protein 1 (PABPC1) and eukaryotic translation initiation factor 4A/G (elF4A/G) to abolish the formation of the translation initiation complex, while tsRNA with the same sequence but without the \(\Psi\)8 modification cannot [158]. However, tsRNA with distinct sequences and secondary structures can be recognized by the same RNA binding proteins under different circumstances. As mentioned above, Y-box binding protein 1 (YBX1) can bind with tsRNA with intermolecular RNA G4 to facilitate the assembly of stress granules in regulating overall cellular translation. Evidence also shows that YBX1 can recognize and bind to inner'tsRNA derived from the anticodon loop of tRNA\({}^{\text{Gly(TCC)}}\), tRNA\({}^{\text{Glu(TTC)}}\), tRNA\({}^{\text{Asp(GTC)}}\), etc., with the SCUBYC motif under hypoxia stress [159]. These hypoxia stress-induced tsRNAs competitively bind to YBX1 by displacing mRNA, resulting in the destabilization of pro-oncogenic transcripts and the repression of tumor metastasis under hypoxia. However, the mechanism of tsRNA-YBX1 recognition and binding still needs further investigation.