Long noncoding RNAs (lncRNAs) - transcripts longer than 200 nucleotides with little evidence of protein coding potential - have been implicated in a wide range of human neurological disorders including cancer, developmental delay, schizophrenia and Alzheimer's disease. While the mammalian genome has been discovered to transcribe many thousands of lncRNAs, very few lncRNAs have been characterized in terms of in vivo function and molecular mechanism. In a genome-wide analysis of lncRNAs in adult ventricular- subventricular zone (V-SVZ) neurogenesis, we identified a novel lncRNA transcript named Pinky (Pnky). We have recently demonstrated that Pnky regulates the production of neurons from NSCs of the embryonic and postnatal brain. Pnky is a neural-specific, nuclear lncRNA transcript. In the V-SVZ neurogenic lineage, Pnky is expressed in NSCs and becomes downregulated during neuronal differentiation. In postnatal V-SVZ NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Mass spectrometry, Western blot, and RNA immunoprecipitation analysis indicates that Pnky physically interacts with PTBP1, a known regulator of neurogenesis, brain tumors, direct cell reprogramming, and RNA splicing. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. We have since generated a Pnky conditional knockout (Pnky-cKO) mouse, and this genetic model of Pnky-deficiency phenocopied Pnky knockdown both in vitro and in vivo. The overall goal of the proposed work is to understand the in vivo function and mechanism of Pnky.
Aim 1 is to determine the role of Pnky in adult V-SVZ neurogenesis by studying Pnky-deficiency and Pnky transgenic expression in vivo. Preliminary Data, our expertise in V-SVZ biology, and the use of multiple, complementary approaches for manipulating Pnky expression support the feasibility of Aim 1.
Aim 2 is to determine the mechanism(s) by which Pnky regulates neurogenesis. Whether Pnky and PTBP1 functionally interact will be investigated with the analysis of biological phenotypes, transcriptome changes, and RNA-protein interactions. The discovery of additional factors that interact with Pnky will provide the basis for investigating other potential lncRNA mechanisms. In addition to Preliminary Data, collaborations with Dr. Aaron Diaz (bioinformatics), Dr. Nevan Krogan (mass spectrometry), Dr. Seth Blackshaw (protein microarrays), and Dr. Hiten Madhani (RNA splicing, RNA-protein interactions) support the feasibility of Aim 2. Such knowledge of lncRNA developmental and mechanistic function will provide critical insight into how lncRNAs can underlie neurological disease and may inform the development of lncRNAs as therapeutic targets.

Public Health Relevance

The human genome encodes many thousands of long noncoding RNA (lncRNA) molecules, and emerging evidence indicates that abnormal production of specific lncRNAs may underlie some of the most devastating human diseases such as cancer, Alzheimer's disease, and schizophrenia. Discovering how lncRNAs regulate stem cell populations is important to understanding how they may cause to human diseases. Furthermore, understanding how lncRNAs function at the molecular level is critical to the development of treatments that utilize lncRNAs as therapeutic targets.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
Project #
Application #
Study Section
Neurogenesis and Cell Fate Study Section (NCF)
Program Officer
Riddle, Robert D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Francisco
Schools of Medicine
San Francisco
United States
Zip Code
Andersen, Rebecca E; Lim, Daniel A (2018) Forging our understanding of lncRNAs in the brain. Cell Tissue Res 371:55-71
Liu, S John; Lim, Daniel A (2018) Modulating the expression of long non-coding RNAs for functional studies. EMBO Rep 19:
Cho, Seung Woo; Xu, Jin; Sun, Ruping et al. (2018) Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element. Cell 173:1398-1412.e22
Nowakowski, Tomasz J; Bhaduri, Aparna; Pollen, Alex A et al. (2017) Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex. Science 358:1318-1323
Delgado, Ryan N; Lim, Daniel A (2017) Maintenance of Positional Identity of Neural Progenitors in the Embryonic and Postnatal Telencephalon. Front Mol Neurosci 10:373
Liu, S John; Horlbeck, Max A; Cho, Seung Woo et al. (2017) CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355:
Ramos, Alexander D; Attenello, Frank J; Lim, Daniel A (2016) Uncovering the roles of long noncoding RNAs in neural development and glioma progression. Neurosci Lett 625:70-9
Liu, Siyuan John; Nowakowski, Tomasz J; Pollen, Alex A et al. (2016) Single-cell analysis of long non-coding RNAs in the developing human neocortex. Genome Biol 17:67
Müller, Sören; Liu, Siyuan John; Di Lullo, Elizabeth et al. (2016) Single-cell sequencing maps gene expression to mutational phylogenies in PDGF- and EGF-driven gliomas. Mol Syst Biol 12:889