A bifunctional, moonlighting RNA coordinates pathways to regulate human embryonic development [scRNA-seq]

Early embryogenesis and cellular differentiation require complex choreography between nuclear and cytoplasmic pathways. Through integrative analysis and a short open reading frame functional screen, sORF-seq, we identified lncPRESS1, a bifunctional long-non-coding RNA that also encodes a microprotein. In the nucleus, lncPRESS1 binds BRG1 to regulate chromatin remodeling and modulate the expression of pluripotency and differentiation genes. In the cytoplasm, lncPRESS1 is translated into a microprotein that binds to IFT57, regulating ciliogenesis and SHH signaling pathways to control lineage commitment. Together, these results reveal a novel mechanism where the dual functions of lncPRESS1 simultaneously coordinate chromatin remodeling and signaling pathways. This coordination co-regulates the initial developmental decisions specifying neuroectoderm and mesoderm fates during human embryonic development, underscoring the underappreciated complexity of multifunctional RNAs. Overall design: The lncPRESS1 WT, KO, and ?ATG hESCs were pretreated with 3.25 uM of CHIR99021 for 24 hours and then 0.5 uM of CHIR99021 for 72 hours to develop 3D gastruloids. These gastruloids were dissociated into single cells using ACCUTASE and then sent for scRNA-seq analysis.

早期胚胎发生与细胞分化过程，依赖于核与细胞质通路间的复杂协同调控。本研究通过整合分析与短开放阅读框（short open reading frame, sORF）功能筛选技术sORF-seq，鉴定出lncPRESS1——一种兼具双重功能的长链非编码RNA（long non-coding RNA, lncRNA），同时亦可编码一种微蛋白。
在细胞核中，lncPRESS1可结合BRG1，调控染色质重塑过程，并调节多能性相关基因与分化相关基因的表达水平。在细胞质中，lncPRESS1可被翻译为一种微蛋白，该蛋白结合IFT57，通过调控纤毛发生与音猬因子（Sonic Hedgehog, SHH）信号通路，控制细胞谱系定型。
综上，本研究结果揭示了一种全新的调控机制：lncPRESS1的双重功能可同时协同调控染色质重塑与信号通路。这一协同调控过程共同调控了人类胚胎发育过程中神经外胚层与中胚层命运决定的早期发育决策，凸显了多功能RNA此前未被充分认知的复杂性。
整体实验设计：将lncPRESS1野生型（wild type, WT）、敲除型（knockout, KO）及ATG缺失型（ΔATG）人类胚胎干细胞（human embryonic stem cells, hESCs）先用3.25 μM CHIR99021预处理24小时，随后更换为0.5 μM CHIR99021继续培养72小时以诱导形成3D类原肠胚。使用Accutase酶将类原肠胚解离为单细胞悬液，随后进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析。