IMP1-interactive lncRNA regulates human endoderm differentiation via stabilizing WNT targets. IMP1-interactive lncRNA regulates human endoderm differentiation via stabilizing WNT targets

Long noncoding RNAs (lncRNAs) have been shown to play important roles in diverse biological process, including embryonic development and cell differentiation. Extensive studies have revealed the function and mechanism of those lncRNAs adjacent to protein-coding genes (PCGs), but there are relatively fewer reports about the lncRNAs within gene desert, particularly in human early germ layer differentiation. Here based on transcriptome analysis during human definitive endoderm (DE) differentiation, we identified a “desert” lncRNA named CTD-2501M5.1, a cytoplasm-located transcript with no protein-coding gene nearby within the 50 kb genomic region, highly expressed in human definitive endoderm. Depletion of CTD-2501M5.1 by either shRNA or promoter deletion could cause the deficiency of DE differentiation from human pluripotent stem cells (PSCs). The biochemical analysis showed that CTD-2501M5.1 functionally interacted with insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1, also named IMP1), which is necessary for endoderm differentiation demonstrated by loss-of-function assay. We further found depletion of CTD-2501M5.1 could result in reduced WNT signaling activities. More importantly, manipulating WNT activity by chemicals could rescue the phenotype of DE deficiency due to the depletion of CTD-2501M5.1 or IMP1. Mechanistically, CTD-2501M5.1 facilitated the interaction between IMP1 and FZD5 mRNA, stabilizing FZD5 which is required for WNT signaling activation and DE differentiation. Ultimately, our study not only revealed the biological function of a novel desert lncRNA CTD-2501M5.1 in human DE differentiation, but also underlined lncRNA-mediated mRNA stability regulation via IMP1. Overall design: We performed RNA-seq using hESC-derived wildtype, lncRNA CTD-2501M5.1-KO and IMP1-KO definitive endoderm cells. IMP1 RIPseq was performed in wildtype and CTD-2501M5.1-KO definitive endoderm cell.

长链非编码RNA（long noncoding RNAs，lncRNAs）已被证实可参与多种生物学过程，包括胚胎发育与细胞分化。既往大量研究已阐明蛋白编码基因（protein-coding genes，PCGs）邻近的lncRNAs的功能与作用机制，但针对基因沙漠（gene desert）区域内lncRNAs的相关报道相对较少，在人类早期胚层分化领域尤为如此。本研究基于人类定形内胚层（definitive endoderm，DE）分化过程中的转录组分析，鉴定出一款名为CTD-2501M5.1的“沙漠”型lncRNA：该转录本定位于细胞质，其50kb基因组范围内无邻近蛋白编码基因，且在人类定形内胚层中呈高表达水平。通过短发夹RNA（short hairpin RNA，shRNA）或启动子缺失技术敲除CTD-2501M5.1，可导致人类多能干细胞（pluripotent stem cells，PSCs）向定形内胚层的分化出现缺陷。生化分析结果显示，CTD-2501M5.1可与胰岛素样生长因子2 mRNA结合蛋白1（insulin-like growth factor 2 mRNA binding protein 1，IGF2BP1，又称IMP1）发生功能性互作；经功能缺失实验证实，该蛋白对于内胚层分化至关重要。进一步研究发现，敲低CTD-2501M5.1会降低WNT信号通路的活性。更为关键的是，通过小分子化合物调控WNT信号通路活性，可挽救因CTD-2501M5.1或IMP1缺失导致的定形内胚层分化缺陷。机制层面，CTD-2501M5.1可促进IMP1与FZD5 mRNA的结合，进而稳定FZD5转录本——而FZD5是WNT信号通路激活与定形内胚层分化所必需的因子。本研究不仅揭示了新型沙漠型lncRNA CTD-2501M5.1在人类定形内胚层分化中的生物学功能，还阐明了lncRNA通过IMP1介导mRNA稳定性调控的机制。
整体实验设计：我们对人类胚胎干细胞（human embryonic stem cells，hESCs）来源的野生型、lncRNA CTD-2501M5.1基因敲除（knockout，KO）及IMP1基因敲除定形内胚层细胞开展了RNA测序（RNA-seq）。同时，在野生型与CTD-2501M5.1基因敲除的定形内胚层细胞中进行了IMP1 RNA结合蛋白免疫沉淀测序（RNA immunoprecipitation sequencing，RIP-seq）。