Functional long noncoding RNAs in neural development

神经发育中的功能性长非编码RNA

• 批准号: 10530928
• 负责人: DANIEL A LIM
• 金额: $ 63.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10530928
• 关键词: Alzheimer' s Disease; Bacterial Artificial Chromosomes; Biological Process; Brain; CRISPR interference; Cell Proliferation; Code; Communities; Coupled; Data; Development; Developmental Delay Disorders; Disease; Drug Targeting; Genes; Genetic study; Genome; Goals; Human; Human Cell Line; Human Genome; In Vitro; Individual; Knowledge; Malignant Neoplasms; Maps; Mental disorders; Messenger RNA; Molecular; Molecular Genetics; Names; Nature; Neurodegenerative Disorders; Neurons; Organoids; Outcome; Pathway interactions; Polypyrimidine Tract-Binding Protein; Production; RNA Splicing; Research; Role; Schizophrenia; Structure; Testing; Transcript; Transgenes; Transplantation; Untranslated RNA; Work; base; cell type; conditional knockout; data resource; epigenomics; genome-wide; human disease; human stem cells; in vivo; induced pluripotent stem cell; insight; knock-down; nerve stem cell; nervous system disorder; neurodevelopment; neurogenesis; novel; relating to nervous system; single-cell RNA sequencing; therapeutic target; therapy development; transcriptome; transcriptomics; transdifferentiation

ABSTRACT Long noncoding RNAs (lncRNAs) have been implicated in a wide range of human neurological disorders including cancer, developmental delay, psychiatric and neurodegenerative disease. While it is known that the brain is enriched in specific lncRNAs, relatively few have been characterized in terms of function and molecular mechanism. Our long-term goal is to understand the function and molecular mechanisms of lncRNAs in neurodevelopment. Such fundamental knowledge is critical to understanding how this large aspect of the noncoding genome regulates brain development and disease. We have taken two approaches for the study of lncRNA function. The first approach is a “traditional” molecular-genetic study of a brain-specific, evolutionarily conserved lncRNA that is a potent regulator of neural stem cells (NSCs). In previous studies, we identified a novel lncRNA transcript that we named Pnky (Pou3f2 intergenic non-koding). In cultured NSCs, either Pnky transcript knockdown or Pnky conditional knockout (Pnky-cKO) increases neuronal production by ~4-fold. Pnky is required for proper cortical neurogenesis in vivo, and the expression of Pnky from a BAC transgene (BAC-Pnky) fully rescues Pnky-deletion – including at the level of the transcriptome – indicating that this lncRNA functions in trans. Pnky interacts with the splicing regulator PTBP1 (Polypyrimidine tract binding protein 1) – a critical regulator of neurogenesis from NSCs – and Pnky appears to function in the same molecular pathway as PTBP1. Preliminary Studies demonstrate that Pnky folds into a compact, monodisperse state that contains intricate structures including a pseudoknot, which is a structural module known to have important function in noncoding RNAs. Given these data, we hypothesize that Pnky contains functional structural modules and regulates the function of PTBP1. Our second approach is to use systematic functional screens to discover key principles of lncRNA genome function. In Preliminary Studies, we used CRISPRi to screen in parallel 10,671 lncRNA and 18,905 mRNA genes for roles in the neural induction of NSCs from human induced pluripotent stem cells (iPSCs). We also performed CRISPRi perturbation coupled with droplet- based single-cell RNA-Seq (Perturb-Seq) for hundreds of screen hits. Based on results from these systematic studies, our working hypothesis is that functional lncRNAs – in comparison to coding genes – are enriched for roles in “focusing” differentiation to specific neural cell types. To further test this hypothesis, we will study lncRNA function in human brain organoids and extend our screens to analyze neurogenesis. Determining the unique functional roles of lncRNAs and coding genes at genome-scale will have important, broad impact on the interpretation of transcriptomic and epigenomic studies of neurodevelopment. Together, by studying lncRNA function at the level of individual transcripts and also at genome scale, we expect to gain fundamental insights into the function of this large aspect of the noncoding genome.

ABSTRACT Long noncoding RNAs (lncRNAs) have been implicated in a wide range of human neurological disorders including cancer, developmental delay, psychiatric and neurodegenerative disease. While it is known that the brain is enriched in specific lncRNAs, relatively few have been characterized in terms of function and molecular mechanism. Our long-term goal is to understand the function and molecular mechanisms of lncRNAs in neurodevelopment. Such fundamental knowledge is critical to understanding how this large aspect of the noncoding genome regulates brain development and disease. We have taken two approaches for the study of lncRNA function. The first approach is a “traditional” molecular-genetic study of a brain-specific, evolutionarily conserved lncRNA that is a potent regulator of neural stem cells (NSCs). In previous studies, we identified a novel lncRNA transcript that we named Pnky (Pou3f2 intergenic non-koding). In cultured NSCs, either Pnky transcript knockdown or Pnky conditional knockout (Pnky-cKO) increases neuronal production by ~4-fold. Pnky is required for proper cortical neurogenesis in vivo, and the expression of Pnky from a BAC transgene (BAC-Pnky) fully rescues Pnky-deletion – including at the level of the transcriptome – indicating that this lncRNA functions in trans. Pnky interacts with the splicing regulator PTBP1 (Polypyrimidine tract binding protein 1) – a critical regulator of neurogenesis from NSCs – and Pnky appears to function in the same molecular pathway as PTBP1. Preliminary Studies demonstrate that Pnky folds into a compact, monodisperse state that contains intricate structures including a pseudoknot, which is a structural module known to have important function in noncoding RNAs. Given these data, we hypothesize that Pnky contains functional structural modules and regulates the function of PTBP1. Our second approach is to use systematic functional screens to discover key principles of lncRNA genome function. In Preliminary Studies, we used CRISPRi to screen in parallel 10,671 lncRNA and 18,905 mRNA genes for roles in the neural induction of NSCs from human induced pluripotent stem cells (iPSCs). We also performed CRISPRi perturbation coupled with droplet- based single-cell RNA-Seq (Perturb-Seq) for hundreds of screen hits. Based on results from these systematic studies, our working hypothesis is that functional lncRNAs – in comparison to coding genes – are enriched for roles in “focusing” differentiation to specific neural cell types. To further test this hypothesis, we will study lncRNA function in human brain organoids and extend our screens to analyze neurogenesis. Determining the unique functional roles of lncRNAs and coding genes at genome-scale will have important, broad impact on the interpretation of transcriptomic and epigenomic studies of neurodevelopment. Together, by studying lncRNA function at the level of individual transcripts and also at genome scale, we expect to gain fundamental insights into the function of this large aspect of the noncoding genome.