tsRNA-3040b accumulates R-loop to regulate Trim35 transcription, which leads to disordered glycolysis and promotes PAECs proliferation

BACKGROUND: Hypoxia significantly influences the development of pulmonary hypertension (PH). However, the role of transfer RNA-derived small RNAs (tsRNAs) produced by nuclease cleavage on PH, particularly their impact on the proliferation of pulmonary artery endothelial cells (PAECs), remains unclear. METHODS: To detect tsRNA expression, panoramic RNA display by overcoming RNA modification aborted sequencing (PANDORA-seq) sequencing analysis and quantitative polymerase chain reaction (qPCR) were employed. The formation of R-loops between tsRNA and genomic DNA was confirmed through chromatin immunoprecipitation followed by polymerase chain reaction (ChIP-PCR) and Dot-blot analyses. Mouse PAECs and lung tissue were manipulated to either overexpress or inhibit tsRNA-3040b, followed by assessments of cell proliferation, RT-qPCR, and enzyme activity assays on three key glycolytic rate-limiting enzymes. Molecular docking, immunofluorescence and endogenous coprecipitation were used to demonstrate the colocalization of Trim35 and Wnt3a. RESULTS: The expression of tsRNA-Asp-GTC-3040b (termed tsRNA-3040b) was significantly increased in the lung tissue of a hypoxia-induced PH mouse model. By integrating database prediction with RNA sequencing, Trim35 was identified as a downstream target of tsRNA-3040b. ChIP-PCR and Dot-blot analyses using S9.6 indicated that tsRNA-3040b promoted R-loops in the genomic DNA of Trim35, thus inhibiting its transcription. Further investigation revealed that the Trim35 affected glucose metabolism levels through ubiquitinated substrate Wnt3a. Ultimately, it was elucidated that the tsRNA-3040b-Trim35-Wnt3a-glucose metabolism signaling pathway exacerbated the progression of PH. CONCLUSIONS: This study highlights the role of tsRNA-3040b in promoting PH by influencing glucose metabolism processes. These results offer a new approach to treating PH and suggest that tsRNA-3040b could serve as a potential target for diagnosing PH and related conditions.