AbstractLong noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Here, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated during pancreas development. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for endocrine cell differentiation. However, deletion of LINC00261 diminishes generation of insulin+ endocrine cells, in a manner independent of the nearby TF FOXA2. Systematic deletion of each of LINC00261’s seven poorly conserved microproteins shows that the RNA, rather than the microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs into recently evolved microproteins during human pancreas development and provides a blueprint for dissection of their coding and noncoding roles.Graphical AbstractHighlightsExtensive lncRNA translation and microprotein production during human pancreas developmentA small-scale loss-of-function screen shows most translated lncRNAs are dispensableLINC00261 is highly translated and regulates endocrine cell differentiationDeleting LINC00261’s evolutionary young microproteins reveals no essential roles
Understanding human fetal pancreas development using subpopulation sorting, RNA sequencing and single-cell profiling