IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions (RIP-Seq). IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions (RIP-Seq)

The insulin-like growth factor 2 mRNA binding protein (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization, and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates. However, the mechanism that regulates its assembly into condensates remains unknown. Here we found, using proteomics, that IGF2BP1 phosphorylation at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulated RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impaired IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increased condensate size and dynamics. Structural approaches showed that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1’s folded domains and these interactions were partially impaired in the Y396E mutant. Our data reveal how phosphorylation modulates low affinity interaction networks in disordered linkers to regulate RNP condensate formation. Overall design: RNA-Seq of HCT116 cells expressing mCherry-IGF2BP1 WT, S181E, Y396E and RQ (R167Q, R168Q, R174Q, R178Q) mutants and RNA immunoprecipitation sequencing (RIP-Seq) of mCherry-IGF2BP1 WT, S181E, Y396E and RQ mutants in control and arsenate stress conditions

胰岛素样生长因子2 mRNA结合蛋白（insulin-like growth factor 2 mRNA binding protein，IGF2BP1）是一类保守的RNA结合蛋白，可调控RNA的稳定性、定位及翻译过程。IGF2BP1是多种核糖核蛋白（ribonucleoprotein，RNP）凝聚体的组成组分，但调控其组装形成RNP凝聚体的分子机制仍有待阐明。本研究通过蛋白质组学分析发现，IGF2BP1无序连接区的S181位点磷酸化水平呈应激依赖性调控。两个无序连接区的磷酸化模拟突变体S181E与Y396E，可在不影响IGF2BP1与RNA结合亲和力的前提下，调控其介导的RNP凝聚体形成。值得注意的是，位于连接区1的S181E突变体在体外及细胞内均会削弱IGF2BP1凝聚体的形成；而位于第二个连接区的Y396E突变体则可增大凝聚体的尺寸并提升其动力学活性。结构生物学分析显示，第一个连接区可通过自身的RGG/RG基序与RNA发生非特异性结合，而该相互作用在S181E突变体中被显著减弱。尤为关键的是，连接区2可与IGF2BP1的折叠结构域发生相互作用，而该相互作用在Y396E突变体中受到部分破坏。本研究数据揭示了磷酸化如何通过调控无序连接区中的低亲和力相互作用网络，进而实现对RNP凝聚体形成的调控。本研究的实验设计如下：对表达mCherry-IGF2BP1野生型（WT）、S181E、Y396E及RQ（R167Q、R168Q、R174Q、R178Q）突变体的HCT116细胞进行RNA测序（RNA-Seq）；同时在正常培养与砷酸盐应激条件下，对表达上述四种mCherry-IGF2BP1变体的细胞进行RNA免疫共沉淀测序（RNA immunoprecipitation sequencing，RIP-Seq）