磷酸化Y14與核酸之液態-液態相分離現象之研究

Abstract

生物分子在時間與空間上的凝聚行為已被視為細胞於DNA雙股斷裂（DSBs）修復過程中的關鍵調控機制。多功能RNA結合蛋白Y14/RBM8A可於DNA損傷部位，透過與RNA介導的交互作用促進非同源性末端接合（NHEJ）的DNA修復途徑。Y14具有帶電的內在無序區域（intrinsically disordered regions, IDRs），提供其進行液-液相分離（liquid-liquid phase separation, LLPS）的結構基礎。其C端RS重複序列經SR蛋白激酶SRPK1磷酸化後，可在鎂離子存在的條件下產生相分離現象。磷酸化後的Y14（pY14）可形成動態凝聚物，並透過不同的交互作用方式與DNA損傷反應（DNA damage response, DDR）因子（如Ku70/80）及核酸分子（包括長鏈非編碼RNA與DNA）共同聚集。非磷酸化Y14與聚（ADP-核糖）（PAR）聚合物的結合親和力高於磷酸化的 Y14，表明 Y14 在被 SRPK1 磷酸化之前就被募集到 DNA 損傷處；隨後磷酸化的Y14在鎂離子存在下形成凝聚物。綜合本研究結果，Y14經磷酸化後所引導的靜電驅動型相分離機制，與鎂離子協同調控，在DNA損傷位置上有助於修復因子的時空性募集與組織。

Spatiotemporal condensation of biomolecules has emerged as a critical mechanism for coordinating the DNA repair process at double-strand breaks (DSBs). The multifunctional RNA-binding protein Y14/RBM8A facilitates non-homologous end joining (NHEJ) through RNA-guided interactions at damage sites. Structurally, Y14 contains charged intrinsically disordered regions (IDRs) that provide the basis for its liquid-liquid phase separation (LLPS). SR protein kinase-1 (SRPK1)-mediated phosphorylation of C-terminal RS dipeptides enables Y14 to undergo magnesium-dependent LLPS in vitro. Phosphorylated Y14 (pY14) condensates accommodate DNA damage response (DDR) factors such as Ku70/80 and nucleic acids such as lncRNA HOTAIRM1. Non-phosphorylated Y14 bound poly(ADP-ribose) (PAR) polymers with a higher affinity than phosphorylated Y14, suggesting that Y14 is recruited to DNA lesions prior to its phosphorylation by SRPK1. Y14 forms condensates after phosphorylation with Mg2+ present. Together, our findings suggested electrostatic interaction-mediated LLPS for recruiting DNA repair factors to Y14 condensates at DNA damage sites.