Effect of fasting and ketogenic diet on hippocampal astrocytes in vivo
Ontology highlight
ABSTRACT: To understand the transcriptional effect of fasting and feeding a ketogenic diet on mouse CNS astrocytes, we performed translating ribosomal affinity purification (TRAP) of mRNAs immunoprecipitated from hippocampus. TRAP mice express a ribosomal epitope tag upon Cre-induced recombination that can be immunoprecipitated following activation. We measured the abundance of actively translating mRNAs from a ribosomal pull-down that came from adult astrocyte (Aldh1l1-Cre)-specific TRAP mice that were subjected to one of three dietary conditions: four weeks of normal chow diet, four weeks of ketogenic diet (high-fat, low-carbohydrate)43, or an 18-hour fast. Immediately following the respective diets, forebrain and hippocampus was harvested from all groups, ribosomes were immunoprecipitated, and actively translating mRNAs in the ribosomes were purified.
Project description:We profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. We also collected the whole embryo translatome using the TRAP protocol and a ubiquitously expressed tagged Rpl10a, and the endothelial transcritome by collecting the GFP+ endothelial cells that had the kdrl promoter driving GFP.
Project description:Translating Ribosome Affinity Purification (TRAP) methods have emerged as a powerful approach to profile actively translated transcripts in specific cell and tissue types. Epitope tagged ribosomal subunits are expressed in defined cell populations and used to pull down ribosomes and their associated mRNAs, providing a snapshot of cell type-specific translation occurring in that space and time. Current TRAP toolkits available to the C. elegans community have been built using multi-copy arrays, randomly integrated in the genome. Here we introduce a Single-copy Knock In Translating Ribosome ImmunoPrecipitation (SKI TRIP) tool kit, a collection of C. elegans strains engineered by CRISPR in which tissue specific expression of FLAG tagged ribosomal subunit protein RPL-22 is driven by cassettes present in single copy from defined sites in the genome. In depth characterization of the SKI TRIP strains and methodology shows that 3xFLAG tagged RPL-22 expressed from its endogenous locus or within defined cell types incorporates into actively translating ribosomes and can be used to efficiently and cleanly pull-down cell type specific transcripts without impacting overall mRNA translation or fitness of the animal. We propose SKI TRIP use for the study of processes that are acutely sensitive to changes in translation, such as aging.
Project description:Neuroanatomical methods enable high-resolution mapping of neural circuitry, but do not allow systematic molecular profiling of neurons based on their connectivity. Here, we report the development of a novel approach for molecularly profiling projective neurons. We show that ribosomes can be labeled with a camelid nanobody raised against GFP and that this system can be engineered to selectively capture translating mRNAs from cells expressing GFP. We generated a transgenic mouse encoding a nanobody-ribosomal protein fusion (Syn-NBL10) and used a retrograde virus (CAV) encoding GFP to immunoprecipitate ribosomes from projection neurons. This enabled us to profile neurons projecting to the nucleus accumbens. The current method provides a new means for profiling neurons based on their projections. Translating mRNAs immunoprecipitated from neurons projecting to the nucleus accumbens. Each Input and IP sample corrspond to a pooled group of 6 mice.
Project description:Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types to control cell fate remains unknown. To determine the magnitude of ribosome heterogeneity and its functional contribution to cell fate specification, we measured ribosomal protein abundance in actively translating ribosomes by quantitative mass spectrometry on a day-by-day basis as human embryonic stem cells differentiate in a step-wise fashion down endoderm and mesoderm lineages. We identified numerous core ribosomal proteins (RPs) as changing significantly in abundance in actively translating ribosomes during cell fate specification, including progressive decreases in ribosome incorporation for several ribosomal proteins during mesoderm differentiation. We further traced ribosome composition changes at the cytoplasmic, whole-cell, and mRNA transcript levels and identified multiple mechanisms regulating actively translating ribosome composition. These findings reveal extensive ribosomal remodeling during differentiation, suggesting that individual ribosomal components may have cell type-specific specialized translation functions.
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits. Ribosomal profiling of large and small polysomal fractions in Drosophila S2 cells to assess translation of smORFs
Project description:We examined here the regulation of gene expression in ventral hippocampus neurons that project to nucleus accumbens (vHPC-NAc) by the transcription factor, ΔFosB. The activity of this circuit is critical to stress-induced social withdrawal. ΔFosB regulates the physiology and behavior of this circuit to produce resilience. Circuit-specific translating ribosomal affinity purification (TRAP) was conducted followed by RNASeq. A strain of floxed Fosb mice (the gene that encodes ΔFosB) was crossed with a ribosomal L10-GFP fusion protein line (Rosa26). To target vHPC-NAc neurons, a retrograde recombinant HSV-Cre was injected into NAc to retrogradely drive expression of GFP and knockout the Fosb gene in vHPC-NAc. Bilateral biopsy punches of vHPC containing GFP-labeled projections were collected, pooled, and processed by TRAP followed by 3rd-generation sequencing of actively translating mRNA. We have now identified Fosb-regulated gene expression in a specific hippocampal-to-accumbens circuit that is important for resilience to stress-induced social withdrawal.
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits.
Project description:NAC (nascent polypeptide-associated complex) post-meiotic heterodimeric αβ-complex promoting chaperone-like cotranslational protein folding on the ribosome and its early role in the birth of nascent proteins is suggested. Here, we demonstrate the presence of specific NAC complex (NACtes) associated with ribosomes in spermatocytes. The RNAseq analysis of mRNAs associated with testis-specific and immunoprecipitated NACtes-carrying ribosomes revealed a preferential association of the latter with mRNAs encoding meiotic and post meiotic proteins. The NACtes ribosomes are also shown to be enriched in mRNAs encoding proteins of central metabolism pathways that have been reported as overexpressed in testes. The specificity of association of NACtes ribosomes with particular mRNA sets was also demonstrated by the observation of significant underrepresentation of abundant mRNAs encoding ubiquitously expressed ribosomal proteins in NACtes-associated ribosomes. At the same time, NACtes ribosomes are enriched in mRNA encoding a testis specific ribosomal protein. These results bring new arguments in favor of “specialized ribosomes hypothesis” proposing a special composition of ribosomes necessary for the control of selected gene expression.