RNA localization is a regulatory mechanism that is conserved from bacteria to mammals. Yet, little is known about the mechanism and the logic that govern the distribution of RNA transcripts within the cell. Here we present a novel organ culture system, which enables the isolation of RNA specifically from NGF dependent re-growing peripheral axons of mouse embryo sensory neurons. In combination with massive parallel sequencing technology, we determine the sub-cellular localization of most transcripts in the transcriptome. We found that the axon is enriched in mRNAs that encode secreted proteins, transcription factors and the translation machinery. In contrast, the axon was largely depleted from mRNAs encoding transmembrane proteins, a particularly interesting finding, since many of these gene products are specifically expressed in the tip of the axon at the protein level. Comparison of the mitochondrial mRNAs encoded in the nucleus with those encoded in the mitochondria, uncovered completely different localization pattern, with the latter much enriched in the axon fraction. This discovery is intriguing since the protein products encoded by the nuclear and mitochondrial genome form large co-complexes. Finally, focusing on alternative splice variants that are specific to axonal fractions, we find short sequence motifs that are enriched in the axonal transcriptome. Together our findings shed light on the extensive role of RNA localization and its characteristics. Overall design: For each RNA sample, Spinal Cords\ DRGs were dissected from 40 E13.5 embryos and cultured for 48H. Total RNA was extracted from whole DRG and Peripheral axons. Poly-A enriched. In duplicates, using GAIIx. Read length - 80nt.
Subcellular transcriptomics-dissection of the mRNA composition in the axonal compartment of sensory neurons.
No sample metadata fields
View SamplesWe used an improved INTACT (Isolation of Nuclei Tagged in A specific Cell Type) technique to isolate RNA from purified nuclei from different neuronal populations of the Drosophila brain. Using RNA-seq, we determined gene expression and A-to-I RNA editing levels at editing sites across nine distinct neuronal sub-populations and a pan-neuronal control. Overall design: We crossed UAS-unc84-2XGFP transgenic flies with 10 different GAL4 drivers (Dh44-GAL4, NPF-GAL4, NPFR-GAL4, Tdc2-GAL4, Crz-GALl4, TH-GAL4, Trh-GAL4, Fru-GAL4, OK107-GAL4, and elav-GAL4), immunoprecipitated tagged nuclei and extracted RNA. Three independent replicates of each each cross were performed to isolate RNA for RNA-sequencing.
Illuminating spatial A-to-I RNA editing signatures within the <i>Drosophila</i> brain.
Subject
View SamplesThe goal of this study is to uncover the changes in the transcriptome of sensory neurons of the liver kinase B1 (LKB1) knockout
Regulation of axonal morphogenesis by the mitochondrial protein Efhd1.
Specimen part
View SamplesThe coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 was implicated in both proliferation and differentiation of multiple regions within the CNS, its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6- retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6- precursors. These alterations were accompanied by co-expression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27kip1 and P27kip2) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks which function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.
Pax6 is required for normal cell-cycle exit and the differentiation kinetics of retinal progenitor cells.
Specimen part
View SamplesThe formation and execution of a productive immune response requires, among many things, the maturation of competent T cells and a robust change in cellular activity upon antigen challenge. Such changes in cellular function require regulated alterations of protein expression. Much work has previously gone into defining the transcriptional changes that regulate protein expression during T cell development and antigen stimulation. Here we describe a parallel pathway of gene regulation that occurs during T cell stimulation, namely alternative splicing. Specifically, we use RNA-Seq to identify 178 exons in 168 genes that exhibit robust changes in inclusion in response to a stimulation of a human T cell line. Interestingly, these signal-responsive genes are enriched for functions related to immune response including, cell trafficking, inflammatory and immune response, immunologic disease and several cell signaling pathways. The vast majority of these genes also exhibit different isoform expression in naive and activated primary T cells. Comparison of the responsiveness of splicing to various stimuli in the cultured and primary T cells reveal important insight into the diversity of signaling pathways that control splicing. Using this data we are able to classify signal-responsive exons into at least three distinct networks. Importantly, we find that each regulatory network is characterized by distinct sequence hallmarks, further suggesting independent regulatory mechanisms. Overall design: We utilize high-throughput RNA sequencing (RNA-Seq) to investigate global changes in alternative splicing in a cultured T cell line and in primary human T cells. We identify 178 genes that are predicted to exhibit robust signal-induced changes in isoform expression in cultured T cells.
Alternative splicing networks regulated by signaling in human T cells.
Specimen part, Subject
View SamplesThe mammalian gastrointestinal tract harbors thousands of bacterial species that include symbionts as well as potential pathogens. The immune responses that limit access of these bacteria to underlying tissue remain poorly defined.
Gammadelta intraepithelial lymphocytes are essential mediators of host-microbial homeostasis at the intestinal mucosal surface.
Specimen part
View SamplesLoss of CLP1 activity results in the accumulation of novel sets of small RNA fragments derived from aberrant processing of tyrosine pre-tRNA. Such tRNA fragments sensitize cells to oxidative stress-induced p53 activation and p53-dependent cell death. Overall design: 2 samples, Wt and Clp1(k/k), no replicates
CLP1 links tRNA metabolism to progressive motor-neuron loss.
Specimen part, Cell line, Subject
View SamplesAbf1 and Rap1 are General Regulatory Factors that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing, and telomere structure in yeast. In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined. We have used microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37 C. We combined this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1. We also identified a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding. Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1. Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1. Finally, we showed for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar "memory effect" that allows continued transcription after loss of Abf1 binding.
Genome-wide analysis of transcriptional dependence and probable target sites for Abf1 and Rap1 in Saccharomyces cerevisiae.
No sample metadata fields
View SamplesExpression profile for undifferentiated F9 Embryonal Carcinoma cell line
Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions.
No sample metadata fields
View SamplesThis study provides a framework describing how magnetic exposure is transduced from the most-plausible molecular-level biosensor (lipid membranes) to cell-level responses that include differentiation toward neural lineages. In addition, SMF provided a stimulus that uncovered new relationships that exist even in the absence of magnetic fields between gangliosides, the time dependent regulation of IL-6 signaling by these glycolipids, and the fate of embryonic cells.
Moderate strength (0.23-0.28 T) static magnetic fields (SMF) modulate signaling and differentiation in human embryonic cells.
No sample metadata fields
View Samples