State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China.
RNA Institute, Wuhan University, Wuhan, 430072, China.
Inflamm Res. 2024 Jul;73(7):1123-1135. doi: 10.1007/s00011-024-01887-4. Epub 2024 May 2.
Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear.
The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells.
HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well.
Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.
针对程序性细胞死亡蛋白 1（PDCD1 或 PD-1）及其配体的免疫疗法显示出了显著的前景，近年来，PD-1 表达的调控机制受到了越来越多的关注。PDCD1 外显子 3 编码跨膜结构域，外显子 3 的缺失会产生 PD-1 的可溶性蛋白同工型（sPD-1），它可以通过与 T 细胞膜表面全长 PD-1 蛋白（flPD-1 或表面 PD-1）竞争来增强免疫反应。然而，PDCD1 外显子 3 跳跃的机制尚不清楚。
使用在线 SpliceAid 程序和小基因表达系统分析参与 PDCD1 外显子 3 剪接事件的潜在剪接因子。通过定点突变技术对 SpliceAid 预测的异质核核糖核蛋白 K（HNRNPK）在外显子 3 上的潜在结合基序进行突变，并通过下拉实验进一步验证。针对 PDCD1 外显子 3 exon 剪接沉默子（ESS）的反义寡核苷酸（ASO）被合成并筛选，以抑制外显子 3 的跳跃。通过半定量逆转录 PCR 分析 PDCD1 外显子 3 的选择性剪接。通过 Western blot 和流式细胞术检测 T 细胞表面 PD-1 的表达。
筛选出 HNRNPK 作为关键剪接因子，促进 PDCD1 外显子 3 跳跃，导致 T 细胞膜上 flPD-1 表达减少，sPD-1 表达增加。从机制上讲，在外显子 3 上鉴定出一个关键的 ESS，它可以与 HNRNPK 蛋白结合，促进外显子 3 跳跃。用 ASO 阻断 ESS 与 HNRNPK 之间的相互作用，显著减少外显子 3 跳跃。重要的是，HNRNPK 也可以促进小鼠 Pdcd1 基因的外显子 3 跳跃。
本研究揭示了 PD-1 表达的一种新的进化保守调控机制。剪接因子 HNRNPK 通过与 PDCD1 外显子 3 上的 ESS 结合，显著促进 PDCD1 外显子 3 跳跃，导致 T 细胞中 flPD-1 表达减少，sPD-1 表达增加。
