K6-Linked Ubiquitylation Signals Reactive Aldehyde-Induced RNA-Protein Crosslink Resolution

Reactive aldehydes induce the formation of RNA-protein crosslinks (RPCs), RPCs in the mRNA stall the ribosome and inhibit translation, RPCs are K6-linked ubiqutylated by RBR-type E3 ligase RNF14, RPCs are resolved in a UBXN1-VCP/p97-dependent manner Proteome-wide iCLIP to detect the distribution of RPC across RNAs; RPCs were introduced with 5 uM 4sU + 0.5 J/cm² UVA (365 nm); protein cleanup with SP3 beads; 2 replicates

活性醛类可诱导RNA-蛋白质交联（RNA-protein crosslinks, RPCs）的形成。信使RNA（messenger RNA, mRNA）上的RPC会阻滞核糖体运转并抑制蛋白质翻译过程；RPC可被RBR型E3泛素连接酶RNF14催化形成K6位泛素化修饰；RPC通过UBXN1-VCP/p97依赖的通路被清除。本数据集采用全蛋白质组单核苷酸分辨率紫外交联免疫沉淀（proteome-wide individual-nucleotide resolution UV crosslinking and immunoprecipitation, iCLIP）技术以检测RPC在各类RNA分子上的分布情况；通过5 μM 4-硫代尿苷（4-thiouridine, 4sU）联合0.5 J/cm²、波长365 nm的长波紫外线（UVA）处理引入RPC；采用SP3磁珠完成蛋白质纯化步骤；实验设置2次生物学重复。