3′-tRF-Cys GCA overexpression in HEK-293 cells alters the global expression profile and modulates cellular processes and pathways

tRNA fragments (tRFs) are small non-coding RNAs generated through specific cleavage of tRNAs and involved in various biological processes. Among the different types of tRFs, the 3′-tRFs have attracted scientific interest due to their regulatory role in gene expression. In this study, we investigated the role of 3′-tRF-Cys GCA , a tRF deriving from cleavage in the T-loop of tRNA CysGCA , in the regulation of gene expression in HEK-293 cells. Previous studies have shown that 3′-tRF-Cys GCA is incorporated into the RISC complex and interacts with Argonaute proteins, suggesting its involvement in the regulation of gene expression. However, the general role and effect of the deregulation of 3′-tRF-Cys GCA levels in human cells have not been investigated so far. To fill this gap, we stably overexpressed 3′-tRF-Cys GCA in HEK-293 cells and performed transcriptomic and proteomic analyses. Moreover, we validated the interaction of this tRF with putative targets, the levels of which were found to be affected by 3′-tRF-Cys GCA overexpression. Lastly, we investigated the implication of 3′-tRF-Cys GCA in various pathways using extensive bioinformatics analysis. Our results indicate that 3′-tRF-Cys GCA overexpression led to changes in the global gene expression profile of HEK-293 cells and that multiple cellular pathways were affected by the deregulation of the levels of this tRF. Additionally, we demonstrated that 3′-tRF-Cys GCA directly interacts with thymopoietin ( TMPO ) transcript variant 1 (also known as LAP2α ), leading to modulation of its levels. In conclusion, our findings suggest that 3′-tRF-Cys GCA plays a significant role in gene expression regulation and highlight the importance of this tRF in cellular processes.