Kim Myeonghwan, Pyo Youngjoon, Hyun Seong-In, Jeong Minseok, Choi Yeon, Kim V Narry
Center for RNA Research, Institute for Basic Science, Seoul, Korea.
School of Biological Sciences, Seoul National University, Seoul, Korea.
Science. 2025 Apr 4;388(6742):eads4539. doi: 10.1126/science.ads4539.
Exogenous messenger RNAs (mRNAs) require cellular machinery for delivery and translation but also encounter inhibitory factors. To investigate their regulation, we performed genome-wide CRISPR screens with in vitro-transcribed mRNAs in lipid nanoparticles (LNPs). Heparan sulfate proteoglycans (HSPGs) and vacuolar adenosine triphosphatase (V-ATPase) were identified as mediators of LNP uptake and endosomal escape, respectively. TRIM25-an RNA binding E3 ubiquitin ligase-emerged as a key suppressor inducing turnover of both linear and circular mRNAs. The endoribonucleases N4BP1 and KHNYN, along with the antiviral protein ZAP, act redundantly in TRIM25-dependent surveillance. TRIM25 specifically targets mRNAs delivered by endosomes, and its RNA affinity increases at acidic pH, suggesting activation by protons released from ruptured endosomes. -methylpseudouridine modification reduces TRIM25's RNA binding, helping RNAs evade its suppressive effect. This study comprehensively maps cellular pathways regulating LNP-mRNAs, offering insights into RNA immunity and therapeutics.
外源性信使核糖核酸（mRNA）需要细胞机制来进行递送和翻译，但也会遇到抑制因子。为了研究它们的调控机制，我们利用脂质纳米颗粒（LNP）包裹的体外转录mRNA进行了全基因组CRISPR筛选。硫酸乙酰肝素蛋白聚糖（HSPG）和液泡型腺苷三磷酸酶（V-ATPase）分别被确定为LNP摄取和内体逃逸的介质。TRIM25——一种RNA结合E3泛素连接酶——成为诱导线性和环状mRNA周转的关键抑制因子。核糖核酸内切酶N4BP1和KHNYN，以及抗病毒蛋白ZAP在TRIM25依赖的监测中发挥冗余作用。TRIM25特异性靶向由内体递送的mRNA，并且其RNA亲和力在酸性pH下增加，这表明它被破裂的内体释放的质子激活。N1-甲基假尿苷修饰降低了TRIM25与RNA的结合，帮助RNA逃避其抑制作用。这项研究全面描绘了调控LNP-mRNA的细胞途径，为RNA免疫和治疗提供了见解。
