Glucose dissociates DDX21 dimers to regulate mRNA processing and promote epidermal differentiation (CLIP-Seq II). Glucose dissociates DDX21 dimers to regulate mRNA processing and promote epidermal differentiation (CLIP-Seq II)

Glucose is an important cellular energy source, however, glucose’s function as a second messenger remains relatively unexplored. Here, we find that glucose binds directly to DDX21 to regulate its function during epidermal differentiation. Specifically, glucose binds to the ATP-binding domain of DDX21 to induce a conformational change and inhibit helicase activity. Glucose binding inhibits the dimerization of DDX21 leading to re-localization from the nucleolus to the nucleoplasm and reassembly of DDX21 into larger protein complexes, increasing its association with splicing factors. This occurs during keratinocyte differentiation, where glucose accumulation is necessary, and results in DDX21 binding to RNA processing proteins and mRNA introns. Consequently, DDX21 regulates the splicing of key differentiation factors and promotes epidermal differentiation in a glucose-dependent manner. These findings reveal a novel mechanism of glucose regulation of cell signaling. Overall design: HEK293T cells were infected with WT or mutant DDX21 to identify DDX21-bound RNAs by CLIP-seq. In addition, three biological replicates of primary human foreskin keratinocytes were used to identify DDX21-bound RNAs by CLIP-seq (easyCLIP variant) in progenitor (non-differentiating) cells in 2D culture. In both cases, cells were infected with lentivirus to express an HA-tagged DDX21 expression construct. CLIP-seq sequencing was performed on a Novaseq, PE150, with ~35 M reads for each sample.

葡萄糖是重要的细胞能源，但其作为第二信使（second messenger）的功能仍未得到充分探索。本研究发现，葡萄糖可直接结合DDX21，调控其在表皮分化过程中的功能。具体而言，葡萄糖结合DDX21的ATP结合结构域（ATP-binding domain），诱导构象变化（conformational change）并抑制解旋酶活性（helicase activity）。葡萄糖结合会抑制DDX21的二聚化（dimerization），使其从核仁（nucleolus）重新定位至核质（nucleoplasm），并重新组装为更大的蛋白质复合物，增强其与剪接因子（splicing factors）的结合。这一过程发生在需要葡萄糖积累的角质细胞（keratinocyte）分化阶段，此时DDX21会结合RNA加工蛋白（RNA processing proteins）与mRNA内含子（mRNA introns）。因此，DDX21调控关键分化因子的剪接，并以葡萄糖依赖的方式促进表皮分化。上述发现揭示了葡萄糖调控细胞信号通路的全新机制。实验整体设计：通过慢病毒（lentivirus）感染HEK293T细胞，使其表达野生型（WT）或突变型DDX21，随后利用CLIP-seq（Cross-Linking Immunoprecipitation and High-throughput Sequencing）鉴定DDX21结合的RNA。此外，我们采用三批生物学重复（biological replicates）的原代人包皮角质细胞（primary human foreskin keratinocytes），在2D培养的增殖（未分化）状态下，通过easyCLIP变体的CLIP-seq技术鉴定DDX21结合的RNA。两组实验中，均通过慢病毒感染细胞以表达HA标签的DDX21重组表达载体。CLIP-seq测序在NovaSeq平台上以PE150模式完成，每个样本的测序读长约为35 M reads。