Project description:Understanding cell diversification from a common genome in metazoans requires single-cell transcriptional analysis. We introduce scFLUENT-seq, a single-cell nascent RNA sequencing method using brief 10-minute metabolic labeling to capture genome-wide transcription. Surprisingly, individual cellsfrom splenic lymphocytes to pluripotent stem cellstranscribe only 0.02~3.1% of the genome, versus >80% in bulk, revealing limited genome engagement and profound cell-type and cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is pervasive and stochastic. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription involves both gene readthrough and independent initiation, while distal intergenic transcription is largely independent of neighboring genes and correlates with increased transcriptional diversity, a hallmark of cellular plasticity. Although global RNA sysnthesis and turnover are coupled in bulk, individual mRNA transcription and decay are poorly coordinated in single cells, suggesting noise-buffering mechanisms. Overall, scFLUENT-seq uncovers complex coding and noncoding transcriptional dynamics that underlie single-cell heterogeneity and state transitions.
Project description:Understanding cell diversification from a common genome in metazoans requires single-cell transcriptional analysis. We introduce scFLUENT-seq, a single-cell nascent RNA sequencing method using brief 10-minute metabolic labeling to capture genome-wide transcription. Surprisingly, individual cellsfrom splenic lymphocytes to pluripotent stem cellstranscribe only 0.02~3.1% of the genome, versus >80% in bulk, revealing limited genome engagement and profound cell-type and cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is pervasive and stochastic. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription involves both gene readthrough and independent initiation, while distal intergenic transcription is largely independent of neighboring genes and correlates with increased transcriptional diversity, a hallmark of cellular plasticity. Although global RNA sysnthesis and turnover are coupled in bulk, individual mRNA transcription and decay are poorly coordinated in single cells, suggesting noise-buffering mechanisms. Overall, scFLUENT-seq uncovers complex coding and noncoding transcriptional dynamics that underlie single-cell heterogeneity and state transitions.
Project description:Understanding cell diversification from a common genome in metazoans requires single-cell transcriptional analysis. We introduce scFLUENT-seq, a single-cell nascent RNA sequencing method using brief 10-minute metabolic labeling to capture genome-wide transcription. Surprisingly, individual cellsfrom splenic lymphocytes to pluripotent stem cellstranscribe only 0.02~3.1% of the genome, versus >80% in bulk, revealing limited genome engagement and profound cell-type and cell-to-cell heterogeneity. Intergenic transcription, especially from heterochromatin, is pervasive and stochastic. Promoter-associated antisense and genic transcription rarely co-occur in the same cell. Proximal intergenic transcription involves both gene readthrough and independent initiation, while distal intergenic transcription is largely independent of neighboring genes and correlates with increased transcriptional diversity, a hallmark of cellular plasticity. Although global RNA sysnthesis and turnover are coupled in bulk, individual mRNA transcription and decay are poorly coordinated in single cells, suggesting noise-buffering mechanisms. Overall, scFLUENT-seq uncovers complex coding and noncoding transcriptional dynamics that underlie single-cell heterogeneity and state transitions.
Project description:Experience-dependent synaptic plasticity refines brain circuits during development. To uncover protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we performed quantitative proteomic analysis of the nascent proteome using improved bio-orthogonal metabolic labeling (BONCAT) to identify candidate plasticity proteins (CPPs) that undergo differential protein synthesis in response to visual conditioning (VC) in Xenopus optic tectum. We identified 83 CPPs that formed strongly connected networks and were annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs using translation blocking morpholinos revealed the requirement of eukaryotic initiation factor 3 subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity of tectal neurons. These results demonstrate that the nascent proteome is dynamic in response to VC and that de novo synthesis of the machinery that regulates gene expression and protein translation is required for experience-dependent structural plasticity.