Interactome of different isoforms of RPL22L1 protein in human glioblastoma cells

Glioblastoma multiforme (GBM) is characterized by an exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here we found that the composition of ribosomes of GBM cells in the tumor core and edge differ due to alternative RNA splicing. The acidic pH in the core switches pre-mRNA splicing of the ribosomal gene RPL22L1 toward the RPL22L1b isoform. This allows cells to survive acidosis, increases stemness and correlates with worse patient outcome. Mechanistically, RPL22L1b promotes RNA splicing by binding to lncMALAT1 in the nucleus, resulting in its degradation. Contrarily, in the tumor edge region, RPL22L1a interacts with ribosomes in cytoplasm and upregulates translation of multiple mRNAs including TP53. We found that RPL22L1 isoform switch is regulated by SRSF4 and identified the compound, that inhibits this process and decreases tumor growth. These findings demonstrate how distinct GBM cell populations arise during tumor growth. Targeting this mechanism may decrease GBM heterogeneity and facilitate therapy. Overall design: We performed precipitation of RNA that interacts with different isoforms of RPL22L1 protein in glioblastoma cells. Ribosomal RNA was depleted and obtained samples were sequenced using HiSeq 2500 (RIPseq experiment).

多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）以极高的肿瘤内异质性为典型特征，但其不同细胞群起源的分子机制仍未明确。本研究发现，肿瘤核心与边缘区域的GBM细胞的核糖体组成因可变RNA剪接而存在差异。肿瘤核心的酸性微环境会诱导核糖体基因RPL22L1的前mRNA剪接偏向RPL22L1b亚型，该亚型可赋予细胞酸中毒耐受性、增强干细胞特性，并与患者不良预后相关。机制层面，RPL22L1b通过在细胞核内结合长链非编码RNA MALAT1（lncMALAT1）并介导其降解，进而促进RNA剪接。与之相反，在肿瘤边缘区域，RPL22L1a与细胞质中的核糖体结合，上调包括TP53在内的多种mRNA的翻译过程。我们证实RPL22L1的亚型剪接转换受剪接因子SRSF4调控，并筛选得到可抑制该过程、减缓肿瘤生长的小分子化合物。上述研究阐明了肿瘤生长过程中不同GBM细胞群的产生机制，靶向该机制或可降低GBM的异质性并助力临床治疗。整体实验设计：我们在胶质母细胞瘤细胞中对与RPL22L1蛋白不同亚型相互作用的RNA进行了富集沉淀。去除核糖体RNA后，采用HiSeq 2500平台对所得样本进行测序，即核糖核酸免疫沉淀测序（RIPseq）实验。