Recent studies expanded our knowledge of diverse pro-survival functions of short non-coding vault RNAs. One of the human vault RNA paralogs, vtRNA1-1, modulates several intracellular processes, including proliferation, apoptosis, autophagy, and drug resistance in various types of human cancer cells. However, protein interaction partners and mechanisms by which vtRNA1-1 levels are controlled within the cells remained elusive. Here, we describe a regulatory process for vtRNA1-1 stabilization mediated by the newly identified interacting proteins, TRIM21 and TRIM25, in human hepatocellular carcinoma (HCC) cells. Depleting TRIM21 or TRIM25 reduced the stability of vtRNA1-1 both in vivo and in vitro. We also identified the responsible sequence of vtRNA1-1 for the stability regulation by TRIM21 and TRIM25 and revealed another critical factor for vtRNA1-1 stability, an NSUN2-mediated methylation at C69 of vtRNA1-1. Consequently, our findings demonstrated that the TRIM proteins govern the stability of vtRNA1-1 depending on its methylation status in HCC cells. Since vtRNA1-1 is crucial for pro-survival characteristics in HCC cells, insight into vtRNA1-1 protein binding partners and the regulation of its stability can impact the development of new anticancer strategies. Author summary: The significance of short non-coding RNA (ncRNA) across various fields of biology has been well established. Among these, vtRNA1-1, one of the four human vault RNA paralogs, is a short but intriguing ncRNA possessing multifaceted roles in regulating cell survival. In this study, we illuminate a novel regulatory mechanism governing vtRNA1-1 stability in human hepatocellular carcinoma (HCC) cells. By analyzing vtRNA-protein complexes, we identify two E3-ligase proteins, namely TRIM21 and TRIM25, which interact with vtRNA1-1 and promote its stability. Additionally, we identified the critical nucleotide sequence required for its stabilization and uncover the role of NSUN2-mediated modification in stabilizing vtRNA1-1. In view of the fact that vtRNA1-1 and these two TRIM proteins are upregulated in liver cancer tissues, it is reasonable to assume that this RNA-protein complex makes an important contribution to liver cancer progression. Furthermore, we demonstrate that depleting TRIM21 or TRIM25 levels in HCC cells enhances sorafenib cytotoxicity, highlighting this vtRNA-protein complex as valuable target for a potential future anticancer strategy. [ABSTRACT FROM AUTHOR]

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