METTL3-dependent m<sup>6</sup>A modification of SNAP29 induces “autophagy-mitochondrial crisis” in the ischemic microenvironment after soft tissue transplantation
Creators: name: Yang, Ningning
name: Lai, Yingying
name: Yu, Gaoxiang
name: Zhang, Xuzi
name: Shi, Jingwei
name: Xiang, Linyi
name: Zhang, Jiacheng
name: Wu, Yuzhe
name: Jiang, Xiaoqiong
name: Zhang, Xuanlong
name: Yang, Liangliang
name: Gao, Weiyang
name: Ding, Jian
name: Wang, Xiangyang
name: Xiao, Jian
name: Zhou, Kailiang
Publisher: name: Taylor & Francis
Year: 2025
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Necrosis at the ischemic distal end of flap transplants increases patients’ pain and economic burden. Reactive oxygen species (ROS) and mitochondrial damage are crucial in regulating parthanatos, but the mechanisms linking disrupted macroautophagic/autophagic flux to parthanatos in ischemic flaps remain unclear. The results of western blotting, immunofluorescence staining, and a proteomic analysis revealed that the autophagic protein SNAP29 was deficient in ischemic flaps, resulting in disrupted autophagic flux, increased ROS-induced parthanatos, and aggravated ischemic flap necrosis. The use of AAV vector to restore SNAP29 <i>in vivo</i> mitigated the disruption of autophagic flux and parthanatos. Additionally, quantification of the total m<sup>6</sup>A level and RIP-qPCR, MeRIP-qPCR, and RNA stability assessments were performed to determine differential <i>Snap29</i> mRNA m<sup>6</sup>A methylation levels and mRNA stability in ischemic flaps. Various <i>in vitro</i> and <i>in vivo</i> tests were conducted to verify the ability of METTL3-mediated m<sup>6</sup>A methylation to promote SNAP29 depletion and disrupt autophagic flux. Finally, we concluded that restoring SNAP29 by inhibiting METTL3 and YTHDF2 reversed the “autophagy-mitochondrial crisis”, defined for the first time as disrupted autophagic flux, mitochondrial damage, mitochondrial protein leakage, and the occurrence of parthanatos. The reversal of this crisis ultimately promoted the survival of ischemic flaps. <b>Abbreviations</b>: AAV = adeno-associated virus; ACTA2/α-SMA = actin alpha 2, smooth muscle, aorta; AIFM/AIF = apoptosis-inducing factor, mitochondrion-associated; ALKBH5 = alkB homolog, RNA demythelase; Baf A1 = bafilomycin A<sub>1</sub>; CQ = chloroquine; DHE = dihydroethidium; ECs = endothelial cells; F-CHP = 5-FAM-conjugated collagen-hybridizing peptide; GO = gene ontology; HUVECs = human umbilical vein endothelial cells; KEGG = Kyoto Encyclopedia of Genes and Genomes; LC-MS/MS = liquid chromatography-tandem mass spectrometry; LDBF = laser doppler blood flow; m<sup>6</sup>A = N6-methyladenosine; MAP1LC3/LC3 = microtubule-associated protein 1 light chain 3; MeRIP = methylated RNA immunoprecipitation; METTL3 = methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit; NAC = N-acetylcysteine; OGD = oxygen glucose deprivation; PAR = poly (ADP-ribose); PARP1 = poly (ADP-ribose) polymerase family, member 1; PECAM1/CD31 = platelet/endothelial cell adhesion molecule 1; ROS = reactive oxygen species; RT-qPCR = reverse transcription quantitative polymerase chain reaction; RIP = RNA immunoprecipitation; SNAP29 = synaptosomal-associated protein 29; SNARE = soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1 = sequestosome 1; SRAMP = sequence-based RNA adenosine methylation site predicting; STX17 = syntaxin 17; TMT = tandem mass tag; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; VAMP8 = vesicle-associated membrane protein 8; WTAP = WT1 associating protein; YTHDF2 = YTH N6-methyladenosine RNA binding protein 2; 3’ UTR = 3ʹ-untranslated region