Pnky lncRNA secondary structure maps identify functional regions that control neurogenesis in neural stem cells

Long noncoding RNA (lncRNA) Pnky is a trans-acting regulator of neural stem cell (NSC) differentiation, but the molecular mechanisms by which Pnky regulates neurogenesis are unknown. A fundamental step toward mechanistic understanding is to determine whether lncRNA structure underlies biological function. Using chemical probing and high-throughput analysis, we determined the secondary structure of Pnky folded in vitro and in cellulo. In vitro-transcribed Pnky RNA adopts a compact, highly structured conformation with evidence of tertiary interactions. In cellulo, Pnky secondary structure is similar to the in vitro conformation. We used locked nucleic acid (LNA) oligonucleotides to interrogate the entire Pnky transcript for function in NSCs and identified regions that when targeted increase neurogenesis-phenocopying Pnky knockdown-without decreasing transcript abundance. Our findings implicate specific structured regions of Pnky in the regulation of neurogenesis and illustrate how structural maps combined with phenotypic data can advance our understanding of lncRNA function.