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I-tRF-Asp promotes vascular remodeling in hypoxic pulmonary hypertension via hnRNPU phase separation which affects TCF7L2 alternative splicing.

ABSTRACT: BACKGROUND: Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary vascular remodeling, primarily driven by excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). While non-coding RNAs such as circular RNAs have been implicated in PH, the role of tRNA-derived small RNA (tsRNA) remains poorly understood. Among them, i-tRF-Asp represents a novel tsRNA whose function in hypoxic PH is unknown. This study aims to investigate whether i-tRF-Asp contributes to PASMC proliferation and vascular remodeling in hypoxic PH. METHODS: To elucidate the functional role of i-tRF-Asp, loss-of-function experiments were conducted in vitro using hypoxic mouse PASMCs (mPASMCs), alongside in vivo studies utilizing both SuHx (Sugen5416+hypoxia) induced pulmonary hypertension models in both adult male C57BL/6 mice and Sprague-Dawley rats. These models employed antisense oligonucleotide inhibitor and adeno-associated virus-mediated silencing. The underlying mechanisms were further explored via RNA immunoprecipitation, liquid-liquid phase separation assays, cell proliferation assessment, western blot, alternative splicing profiling and molecular dynamics simulation. RESULTS: Inhibition of i-tRF-Asp ameliorated hypoxia-induced pulmonary vascular remodeling, primarily through suppressing excessive proliferation of mPASMCs. Further analysis indicated that i-tRF-Asp facilitates liquid–liquid phase separation (LLPS) of hnRNPU. This process mediates selective recruitment of Tcf7l2 pre-mRNA and promotes alternative splicing, resulting in a pro-proliferative Tcf7l2 transcript variant that drives mPASMC proliferation. Furthermore, we have identified that the endonuclease ERN1 can upregulate the expression of i-tRF-Asp. CONCLUSION: The endoribonuclease ERN1 upregulates the expression of i-tRF-Asp, which in turn facilitates mPASMC proliferation and pulmonary vascular remodeling. This process is driven by hnRNPU LLPS-dependent alternative splicing of the long Tcf7l2 transcript isoform.

ORGANISM(S): Mus musculus

PROVIDER: GSE328570 | GEO | 2026/06/03

REPOSITORIES: GEO

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Project description:Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes the conversion of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA), which is critical to prevent auto-inflammatory responses mediated by activation of the type I interferon (IFN) signaling. Here, we define the role of ADAR1-dependent RNA editing in interferon-β(IFNβ) activation and pulmonary artery smooth muscle cell (PASMC) remodeling in pulmonary arterial hypertension (PAH), a devastating disease leading to right heart failure and premature death. Methods: RNA editing levels were analyzed in human pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH (IPAH) patients versus healthy controls. A conditional transgenic line ADAR1SMC-KO was generated by knocking out ADAR1 selectively in α-smooth muscle actin-positive cells, followed by hypoxic exposure to induce PH. Results: PASMCs from IPAH patients displayed decreased levels of ADAR1 mRNA and protein, accompanied by reduced A-to-I editing compared to healthy PASMCs. ADAR1 knockdown in PASMCs upregulated MDA5, PKR, IFN-β and IFN-Stimulated Genes (ISGs). Compared with controls in vivo, hypoxic ADAR1SMC-KO mice developed severe PH, as evidenced by excessive vascular remodeling in distal arterioles and increased vascular leakage resulting in elevated right ventricular systolic pressure and right ventricular hypertrophy. Mechanistically, IFNβ signaling in ADAR1SMC-KO lungs induced the recruitment of inflammatory cells, including macrophages, which in turn enhanced PASMC proliferation and muscularization. Correspondingly, pharmacological treatment with PKR kinase inhibitor Imoxin decreased IFNβ thus attenuating the hypoxia-induced PH phenotype of ADAR1SMC-KO mice. Conclusions: ADAR1-dependent A-to-I RNA editing tempers IFN signaling in PASMCs. Pathologic reduction of ADAR1 in SMCs increased IFNβ activity, macrophage recruitment, and worsened PH, thus demonstrating a direct detrimental role of vascular innate immune responses in PH. Targeting PKR could be the new therapeutic strategy aimed at treating PAH.

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I-tRF-Asp promotes vascular remodeling in hypoxic pulmonary hypertension via hnRNPU phase separation which affects TCF7L2 alternative splicing.

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