Mammalian genomes transcribe thousands of long non-coding RNAs (lncRNAs), RNA molecules longer than 200 nt that do not appear to code for proteins. These diverse transcripts have important roles in fundamental biological processes such as the inactivation of the X chromosome in females, the growth and differentiation of embryonic stem cells, and epigenetic gene regulation. Importantly, lncRNAs have also been implicated in several neurological disorders, including Alzheimer's disease, schizophrenia, autism, and glioma. Despite increasing evidence for the importance of lncRNAs, the in vivo function and molecular mechanisms of lncRNAs remain mostly elusive. To address this knowledge gap, we have recently discovered and begun to characterize a novel, evolutionarily conserved lncRNA termed Pnky that is specifically expressed in the central nervous systems of mice and humans. Pnky is expressed in the neural stem cells (NSCs) of the mouse subventricular zone (SVZ), a brain region that exhibits adult neurogenesis. Remarkably, knockdown of Pnky in cultured SVZ NSCs increases neurogenesis several-fold, and live cell microscopy of single NSCs in culture confirms this finding. RNA-pull down followed by mass spectrometry showed that Pnky physically interacts with PTBP1, a known regulator of neurogenesis that also functions as a splicing factor. Intriguingly, RNA-seq analysis of Pnky knockdown in cultured SVZ NSCs revealed differential splicing of over 150 genes, in addition to differential expression of genes that regulate neurogenesis. We hypothesize that the lncRNA Pnky interacts with PTBP1 to co-regulate a transcriptional program that controls the generation of neurons from neural stem cells.
Aim 1 is to determine the in vivo role of Pnky in SVZ neurogenesis, an ideal in vivo system in which to study neural development. Pnky-deficiency will be targeted to NSCs in the SVZ and cell fate will be determined with lineage tracing methods.
Aim 2 is to understand the molecular mechanisms by which Pnky regulates neurogenesis. To investigate the interaction between Pnky and PTBP1, NSCs with deficiency of Pnky and/or PTBP1 will be analyzed. RNA-seq analysis of these cells will determine the changes in gene expression as well as alternative splicing. In this training program, the candidate is expected to accomplish these Aims by acquiring new knowledge and skills for in vivo neurodevelopmental analysis from the co- sponsors' highly collaborative laboratories that have specific expertise in these areas. Furthermore, the candidate's background in bioinformatics will be broadened and integrated into the study of lncRNAs in neural development, which is an area of research of the primary sponsor. Thus, accomplishment of these Aims will provide new training in terms of knowledge, scientific approaches, and academic skills that are highly relevant to the candidate's long-term career goal of neuroscience research and neuropathology.
Many neurological diseases such as Alzheimer's, schizophrenia, autism, and cancer have been linked to the aberrant functioning of long non-coding RNAs (lncRNAs). We seek to understand the role of a novel lncRNA, Pnky, in neural stem cells, and ask how it interacts with a crucial protein, PTBP1, to control the generation of new neurons in the adult brain. This study will contribute to our knowledge of this diverse class of RNA molecules, potentially revealing drug targets for therapy.
| 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: |
| 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 |
| Ramos, Alexander D; Andersen, Rebecca E; Liu, Siyuan John et al. (2015) The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells. Cell Stem Cell 16:439-447 |
