Much of the human genome does not end up encoding proteins. These noncoding RNA molecules still carry out important functions in many cellular processes, but long noncoding RNAs (lncRNAs) remain a particularly enigmatic class of RNA molecules.
Researchers at UC San Francisco have now determined the structure of Pnky, a lncRNA critical to the production of new neurons. These findings, published in the current issue of Cell Reports, represent the first reported structure of a neuronal lncRNA.
“Not all lncRNAs have structure, but this work shows that Pnky’s structure plays a significant role in how it functions,” said Daniel Lim, MD, PhD, a professor of Neurological Surgery and the co-senior author of the study.
The Lim lab first discovered Pnky in 2015 and identified that it regulates neurogenesis by preventing neural stem cells from differentiating into new neurons. This role, keeping the neural stem cell pool intact, is critical to ensuring that the brain develops properly with the appropriate number of neurons.
“The classical view is that proteins are more important in regulating important neuronal functions, like neurogenesis,” Parna Saha, PhD, a postdoctoral scholar and the co-first author of the study, said. “But here we have a lncRNA with this powerful function.”
In subsequent papers from 2019 and 2024, Lim’s group has demonstrated that depleting Pnky can impact embryonic brain development and behavior in adult mice. But without the 3D structure of the lncRNA molecule, they were still missing a key piece of the puzzle for understanding how it exerts its neurodevelopmental function.
The UCSF scientists teamed up with researchers in the laboratory of Anna Pyle, PhD, a Yale University professor specializing in RNA structural biology. Through this collaboration, they found that the nucleotide sequence of Pnky folds into a highly structured globular molecule with seven distinct regions. It then seems to further fold in on itself to form tertiary interactions.
Pnky is present at very low levels in neural stem cells, with only about 10 to 20 copies of this lncRNA in the nucleus, making it technically challenging to study, says Saha.
But rather than using immortalized cancer cell lines, she and her colleagues devised an experimental approach that enabled them to study its function in primary mouse neural stem cells, a setting that better reflects the neurodevelopmental context.
They then used locked nucleic acid probes to disrupt specific regions of Pnky’s structure without decreasing the abundance of the lncRNA, finding that by targeting certain structural folds of the lncRNA, they could recapitulate the effects of Pnky depletion.
“This now gives us a handle to fine-tune which regions we target to get an output in terms of neurogenesis,” Saha said.
Lim’s team is now trying to figure out how the different modules of Pnky’s structure interact with other RNA molecules or proteins within the nucleus.
“I think we will keep coming back to the structure to see which parts of this lncRNA are important for those interactions,” Saha said.
A similar version of the lncRNA is present in humans, so the Lim lab wants to tease apart the degree of conservation in the structure and function of mouse and human Pnky.
“Working with Dr. Lim and Dr. Saha was an incredible opportunity to join an understanding of RNA structure with that of gene expression in the brain,” Pyle said. "This is only the beginning of an important chapter in RNA neuroscience.”
The work also reinforces the notion that targeting lncRNA structures could have broader translational relevance, and Lim is interested in how increasing neuronal production could be a potential strategy to treat brain injury or disease.
Featured publications:
Saha P, Patel S, Tsao LH, Tavares RC, Choi H, Andersen RE, Pyle AM, Lim DA. Pnky lncRNA secondary structure maps identify functional regions that control neurogenesis in neural stem cells. Cell Rep. 2026 Jun 23;45(6):117451.
Saha P, Andersen RE, Hong SJ, Gil E, Simms J, Choi H, Lim DA. Sex-specific role for the long noncoding RNA Pnky in mouse behavior. Nat Commun. 2024 Aug 12;15(1):6901.
Andersen RE, Hong SJ, Lim JJ, Cui M, Harpur BA, Hwang E, Delgado RN, Ramos AD, Liu SJ, Blencowe BJ, Lim DA. The Long Noncoding RNA Pnky Is a Trans-acting Regulator of Cortical Development In Vivo. Dev Cell. 2019 May 20;49(4):632-642.e7.