Translating Ribosome Affinity Purification Research Articles

Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages

Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.

Regulation of the hippocampal translatome by Apoer2-ICD release

BackgroundApoE4, the most significant genetic risk factor for late-onset Alzheimer’s disease (AD), sequesters a pro-synaptogenic Reelin receptor, Apoer2, in the endosomal compartment and prevents its normal recycling. In the adult brain, Reelin potentiates excitatory synapses and thereby protects against amyloid-β toxicity. Recently, a gain-of-function mutation in Reelin that is protective against early-onset AD has been described. Alternative splicing of the Apoer2 intracellular domain (Apoer2-ICD) regulates Apoer2 signaling. Splicing of juxtamembraneous exon 16 alters the γ-secretase mediated release of the Apoer2-ICD as well as synapse number and LTP, and inclusion of exon 19 ameliorates behavioral deficits in an AD mouse model. The Apoer2-ICD has also been shown to alter transcription of synaptic genes. However, the role of Apoer2-ICD release upon transcriptional regulation and its role in AD pathogenesis is unknown.MethodsTo assess in vivo mRNA-primed ribosomes specifically in hippocampi transduced with Apoer2-ICD splice variants, we crossed wild-type, cKO, and Apoer2 cleavage-resistant mice to a Cre-inducible translating ribosome affinity purification (TRAP) model. This allowed us to perform RNA-Seq on ribosome-loaded mRNA harvested specifically from hippocampal cells transduced with Apoer2-ICDs.ResultsAcross all conditions, we observed ~4,700 altered translating transcripts, several of which comprise key synaptic components such as extracellular matrix and focal adhesions with concomitant perturbation of critical signaling cascades, energy metabolism, translation, and apoptosis. We further demonstrated the ability of the Apoer2-ICD to rescue many of these altered transcripts, underscoring the importance of Apoer2 splicing in synaptic homeostasis. A variety of these altered genes have been implicated in AD, demonstrating how dysregulated Apoer2 splicing may contribute to neurodegeneration.ConclusionsOur findings demonstrate how alternative splicing of the APOE and Reelin receptor Apoer2 and release of the Apoer2-ICD regulates numerous translating transcripts in mouse hippocampi in vivo. These transcripts comprise a wide range of functions, and alterations in these transcripts suggest a mechanistic basis for the synaptic deficits seen in Apoer2 mutant mice and AD patients. Our findings, together with the recently reported AD-protective effects of a Reelin gain-of-function mutation in the presence of an early-onset AD mutation in Presenilin-1, implicate the Reelin/Apoer2 pathway as a target for AD therapeutics.Graphical

Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood–brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

Bridging the gap: parallel profiling of ribosome associated and total RNA species can identify transcriptional regulatory mechanisms of plants in spaceflight

ABSTRACT As plants are an essential component of sustainable life support systems, long-duration space missions will require a sophisticated understanding of plant adaptations to spaceflight and microgravity. For many years, transcriptional profiling of steady state mRNA abundances has been used as measure of plant adaptations to the space environment. However, measured changes in transcript abundances are often not reflected in corresponding changes in the proteome due regulatory processes governing translation. Translating ribosome affinity purification (TRAP) is a technique which selectively targets ribosome bound mRNAs for isolation and downstream sequencing. Comparing profiles of ribosome associated mRNAs with total mRNAs provides insight into the translatome and may more accurately inform on the cellular responses to the spaceflight environment. Toward that goal, this work describes a methodology developed ahead of the APEx-07 flight mission.

Simultaneous measurement of nascent transcriptome and translatome using 4-thiouridine metabolic RNA labeling and translating ribosome affinity purification.

Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation requires nascent RNA synthesis and translation. Analysis of the coordinated regulation of dynamic RNA synthesis and translation is required to determine functional protein production. However, reliable methods for the simultaneous measurement of nascent RNA synthesis and translation at the gene level are limited. Here, we developed a novel method for the simultaneous assessment of nascent RNA synthesis and translation by combining 4-thiouridine (4sU) metabolic RNA labeling and translating ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins. The P-stalk-mediated TRAP (P-TRAP) technique recovered endogenous translating ribosomes, allowing easy translatome analysis of various eukaryotes. We validated this method in mammalian cells by demonstrating that acute unfolded protein response (UPR) in the endoplasmic reticulum (ER) induces dynamic reprogramming of nascent RNA synthesis and translation. Our nascent P-TRAP (nP-TRAP) method may serve as a simple and powerful tool for analyzing the coordinated regulation of transcription and translation of individual genes in various eukaryotes.

Ethanol-induced transcriptional and translational changes in Aldh1l1-Egfp/Rpl10a cortical astrocyte cultures.

The role astrocytes play in brain development and function has garnered greater attention as the diversity of roles they are involved in has become apparent. We have previously shown that ethanol-exposed astrocytes alter neuronal neurite outgrowth in an in vitro co-culture system and that ethanol alters the astrocyte-produced extracellular matrix (ECM) in vitro, with similar alterations in vivo. In this study, we utilized the translating ribosome affinity purification (TRAP) procedure in Aldh1l1-EGFP/Rpl10a transgenic mouse primary cortical astrocyte cultures to transcriptionally and translationally profile the astrocyte response to ethanol. We found a large number of differences between the total RNA pool and the translating RNA pool, indicating that the transcriptional state of astrocytes may not always reflect the translational state of astrocytes. In addition, there was a considerable overlap between ethanol-dysregulated genes in the total RNA pool and the translating RNA pool. Comparisons to published datasets indicate the in vitro model used here is most similar to PD1 or PD7 in vivo cortical astrocytes, and the ethanol-regulated genes showed a significant overlap with models of chronic ethanol exposure in astrocytes, a model of third-trimester ethanol exposure in the hippocampus and cerebellum, and an acute model of ethanol exposure in the hippocampus. These findings will further our understanding of the effects of ethanol on astrocyte gene expression and protein translation and how these changes may alter brain development and support the use of in vitro astrocyte cultures as models of neonatal astrocytes.

Cell-specific RNA profiling reveals host genes expressed in Arabidopsis cells haustoriated by downy mildew.

The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infects Arabidopsis (Arabidopsis thaliana) by forming structures called haustoria inside host cells. Previous transcriptome analyses have revealed host genes are specifically induced during infection; however, RNA profiling from whole infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis and H. arabidopsidis at the cellular level, we devised a translating ribosome affinity purification (TRAP) system using two high-affinity binding proteins, colicin E9 and Im9 (immunity protein of colicin E9), applicable to pathogen-responsive promoters, thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing insights into the Arabidopsis-downy mildew interaction. We propose that our protocol for profiling cell-specific transcripts will apply to several stimulus-specific contexts and other plant-pathogen interactions.

Abstract 219: Translational Profiling Of Embryonic And Adult-derived Macrophages Exposed To Hypoxia

Cardiac-resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Post-transcriptional regulation at the levels of mRNA stability and translation may override transcriptional signals in response to hypoxia. We hypothesized that profiling of actively translated mRNAs in BMDM and FLDM after exposure to hypoxia would uncover functional differences in gene expression between these macrophage subsets. We used a translating ribosome affinity purification (TRAP) assay coupled with RNA-seq and validation by qRT-PCR, flow cytometry and immunoblotting to identify translated transcripts in macrophages exposed to hypoxia. We identified non-overlapping transcripts with increased translation in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2). Induction of most transcripts, including those encoding enzymes associated with glycolysis, was accompanied by increased translation. However, induction of the mRNA encoding complement receptor C5ar1 was accompanied by reduced translation efficiency. Furthermore, we determined the effects of deletion of the RNA-binding protein HuR on induction and translation of transcripts in response to hypoxia. Overall, these findings define translational differences in macrophage subset gene expression programs, highlighting potential macrophage-directed therapeutic targets in ischemic myocardium.

Early Life Antibiotic Exposures Inhibits Postnatal Enteric Nervous System Maturation and Alters Neonatal Neuroimmune Interactions

Abstract The enteric nervous system (ENS) regulates intestinal homeostasis via interactions with smooth muscle, epithelium, and immune cells. To assess the impact of early life antibiotics on postnatal ENS development, we performed neuron specific translating ribosome affinity purification to isolate RNA from control and antibiotic exposed enteric neurons for RNA sequencing. Colonic enteric neurons from antibiotic exposed pre-weaning mice expressed significantly less mature neuron markers, and acquired a more inflammatory cytokine signaling signature (e.g. IL18, MCP-1, TLR4). Immunofluorescence staining and ELISA revealed increased enteric neuron density in antibiotic exposed colons, yet reduced expression of neuronal markers including Tuj1 (neuron specific tubulin), synaptophysin (synapse marker) and acetylcholine. Notably, antibiotics did not affect ENS development in germ free mice. By confocal microscopy, antibiotic exposure appeared to reduce phagocytosis of neuronal cell bodies by macrophages, but phagocytosis of synapses by macrophages was increased. Flow cytometry revealed that antibiotic exposure reduced the proportion of macrophages expressing the “M2” marker CD163 compared to controls. This observation is reminiscent of Hirschsprung disease (HSCR), a congenital absence of the ENS from the distal intestine, which causes intestinal macrophages to adopt a pro-inflammatory phenotype and predisposes patients for HSCR-associated enterocolitis secondary to decreased cholinergic signaling. These data suggest that early life antibiotic exposure disrupts how macrophages participate in postnatal ENS development in a microbiota dependent mechanism. NIH NIAID U01AI131342, NIH NIDDK P30DK052574, T32DK077653, NIH T32HD43010 American Academy of Pediatrics Marshall Klaus 2022

Effects of alcohol and PARP inhibition on RNA ribosomal engagement in cortical excitatory neurons.

We report on the effects of ethanol (EtOH) and Poly (ADP-ribose) polymerase (PARP) inhibition on RNA ribosomal engagement, as a proxy for protein translation, in prefrontal cortical (PFC) pyramidal neurons. We hypothesized that EtOH induces a shift in RNA ribosomal-engagement (RE) in PFC pyramidal neurons, and that many of these changes can be reversed using a PARP inhibitor. We utilized the translating ribosome affinity purification (TRAP) technique to isolate cell type-specific RNA. Transgenic mice with EGFP-tagged Rpl10a ribosomal protein expressed only in CaMKIIα-expressing pyramidal cells were administered EtOH or normal saline (CTL) i.p. twice a day, for four consecutive days. On the fourth day, a sub-group of mice that received EtOH in the previous three days received a combination of EtOH and the PARP inhibitor ABT-888 (EtOH + ABT-888). PFC tissue was processed to isolate both, CaMKIIα pyramidal cell-type specific ribosomal-engaged RNA (TRAP-RNA), as well as genomically expressed total-RNA from whole tissue, which were submitted for RNA-seq. We observed EtOH effects on RE transcripts in pyramidal cells and furthermore treatment with a PARP inhibitor "reversed" these effects. The PARP inhibitor ABT-888 reversed 82% of the EtOH-induced changes in RE (TRAP-RNA), and similarly 83% in the total-RNA transcripts. We identified Insulin Receptor Signaling as highly enriched in the ethanol-regulated and PARP-reverted RE pool and validated five participating genes from this pathway. To our knowledge, this is the first description of the effects of EtOH on excitatory neuron RE transcripts from total-RNA and provides insights into PARP-mediated regulation of EtOH effects.

Molecular Profiling of VGluT1 AND VGluT2 Ventral Subiculum to Nucleus Accumbens Shell Projections.

The nucleus accumbens shell is a critical node in reward circuitry, encoding environments associated with reward. Long-range inputs from the ventral hippocampus (ventral subiculum) to the nucleus accumbens shell have been identified, yet their precise molecular phenotype remains to be determined. Here we used retrograde tracing to identify the ventral subiculum as the brain region with the densest glutamatergic (VGluT1-Slc17a7) input to the shell. We then used circuit-directed translating ribosome affinity purification to examine the molecular characteristics of distinct glutamatergic (VGluT1, VGluT2-Slc17a6) ventral subiculum to nucleus accumbens shell projections. We immunoprecipitated translating ribosomes from this population of projection neurons and analysed molecular connectomic information using RNA sequencing. We found differential gene enrichment across both glutamatergic projection neuron subtypes. In VGluT1 projections, we found enrichment of Pfkl, a gene involved in glucose metabolism. In VGluT2 projections, we found a depletion of Sparcl1 and Dlg1, genes known to play a role in depression- and addiction-related behaviours. These findings highlight potential glutamatergic neuronal-projection-specific differences in ventral subiculum to nucleus accumbens shell projections. Together these data advance our understanding of the phenotype of a defined brain circuit.

Decoding the molecular crosstalk between grafted stem cells and the stroke-injured brain

Stem cell therapy shows promise for multiple disorders; however, the molecular crosstalk between grafted cells and host tissue is largely unknown. Here, we take a step toward addressing this question. Using translating ribosome affinity purification (TRAP) with sequencing tools, we simultaneously decode the transcriptomes of graft and host for human neural stem cells (hNSCs) transplanted into the stroke-injured rat brain. Employing pathway analysis tools, we investigate the interactions between the two transcriptomes to predict molecular pathways linking host and graft genes; as proof of concept, we predict host-secreted factors that signal to the graft and the downstream molecular cascades they trigger in the graft. We identify a potential host-graft crosstalk pathway where BMP6 from the stroke-injured brain induces graft secretion of noggin, a known brain repair factor. Decoding the molecular interplay between graft and host is a critical step toward deciphering the molecular mechanisms of stem cell action.

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

Neither GLP-1 receptors nor GFRAL neurons are required for aversive or anorectic response to DON (vomitoxin).

Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grains, promotes nausea, emesis and anorexia. With DON exposure, circulating levels of intestinally derived satiation hormones, including glucagon-like peptide 1 (GLP-1) are elevated. To directly test whether GLP-1 signaling mediates the effects of DON, we examined the response of GLP-1 or GLP-1R-deficient mice to DON injection. We found comparable anorectic and conditioned taste avoidance learning responses in GLP-1/GLP-1R deficient mice compared to control littermates, suggesting that GLP-1 is not necessary for the effects of DON on food intake and visceral illness. We then used our previously published data from translating ribosome affinity purification with RNA sequencing (TRAP-seq) analysis of area postrema neurons that express the receptor for the circulating cytokine growth differentiation factor (GDF15), growth differentiation factor a-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, calcium sensing receptor (CaSR), is heavily enriched in GFRAL neurons. Given that GDF15 potently reduces food intake and can cause visceral illness by signaling through GFRAL neurons, we hypothesized that DON may also signal by activating CaSR on GFRAL neurons. Indeed, circulating GDF15 levels are elevated after DON administration but both GFRAL knockout and GFRAL neuron-ablated mice exhibited similar anorectic and conditioned taste avoidance responses compared to WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.

Mu-opioid receptor-expressing neurons in the paraventricular thalamus modulate chronic morphine-induced wake alterations

Disrupted sleep is a symptom of many psychiatric disorders, including substance use disorders. Most drugs of abuse, including opioids, disrupt sleep. However, the extent and consequence of opioid-induced sleep disturbance, especially during chronic drug exposure, is understudied. We have previously shown that sleep disturbance alters voluntary morphine intake. Here, we examine the effects of acute and chronic morphine exposure on sleep. Using an oral self-administration paradigm, we show that morphine disrupts sleep, most significantly during the dark cycle in chronic morphine, with a concomitant sustained increase in neural activity in the Paraventricular Nucleus of the Thalamus (PVT). Morphine binds primarily to Mu Opioid Receptors (MORs), which are highly expressed in the PVT. Translating Ribosome Affinity Purification (TRAP)-Sequencing of PVT neurons that express MORs showed significant enrichment of the circadian entrainment pathway. To determine whether MOR + cells in the PVT mediate morphine-induced sleep/wake properties, we inhibited these neurons during the dark cycle while mice were self-administering morphine. This inhibition decreased morphine-induced wakefulness but not general wakefulness, indicating that MORs in the PVT contribute to opioid-specific wake alterations. Overall, our results suggest an important role for PVT neurons that express MORs in mediating morphine-induced sleep disturbance.

Atlas of phosphoinositide signatures in the retina identifies heterogeneity between cell types.

Phosphoinositides (PIPs) are a family of minor acidic phospholipids in the cell membrane. Phosphoinositide (PI) kinases and phosphatases can rapidly convert one PIP product into another resulting in the generation of seven distinct PIPs. The retina is a heterogeneous tissue composed of several cell types. In the mammalian genome, around 50 genes encode PI kinases and PI phosphatases; however, there are no studies describing the distribution of these enzymes in the various retinal cell types. Using translating ribosome affinity purification, we have identified the in vivo distribution of PI-converting enzymes from the rod, cone, retinal pigment epithelium (RPE), Müller glia, and retinal ganglion cells, generating a physiological atlas for PI-converting enzyme expression in the retina. The retinal neurons, rods, cones, and RGCs, are characterized by the enrichment of PI-converting enzymes, whereas the Müller glia and RPE are characterized by the depletion of these enzymes. We also found distinct differences between the expression of PI kinases and PI phosphatases in each retinal cell type. Since mutations in PI-converting enzymes are linked to human diseases including retinal diseases, the results of this study will provide a guide for what cell types are likely to be affected by retinal degenerative diseases brought on by changes in PI metabolism.

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

The function of lactate dehydrogenase A in retinal neurons: implications to retinal degenerative diseases.

The postmitotic retina is highly metabolic and the photoreceptors depend on aerobic glycolysis for an energy source and cellular anabolic activities. Lactate dehydrogenase A (LDHA) is a key enzyme in aerobic glycolysis, which converts pyruvate to lactate. Here we show that cell-type-specific actively translating mRNA purification by translating ribosome affinity purification shows a predominant expression of LDHA in rods and cones and LDHB in the retinal pigment epithelium and Müller cells. We show that genetic ablation of LDHA in the retina resulted in diminished visual function, loss of structure, and a loss of dorsal-ventral patterning of the cone-opsin gradient. Loss of LDHA in the retina resulted in increased glucose availability, promoted oxidative phosphorylation, and upregulated the expression of glutamine synthetase (GS), a neuron survival factor. However, lacking LDHA in Müller cells does not affect visual function in mice. Glucose shortage is associated with retinal diseases, such as age-related macular degeneration (AMD), and regulating the levels of LDHA may have therapeutic relevance. These data demonstrate the unique and unexplored roles of LDHA in the maintenance of a healthy retina.

Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. Invivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

Early-life exercise primes the murine neural epigenome to facilitate gene expression and hippocampal memory consolidation

Aerobic exercise is well known to promote neuroplasticity and hippocampal memory. In the developing brain, early-life exercise (ELE) can lead to persistent improvements in hippocampal function, yet molecular mechanisms underlying this phenomenon have not been fully explored. In this study, transgenic mice harboring the “NuTRAP” (Nuclear tagging and Translating Ribosome Affinity Purification) cassette in Emx1 expressing neurons (“Emx1-NuTRAP” mice) undergo ELE during adolescence. We then simultaneously isolate and sequence translating mRNA and nuclear chromatin from single hippocampal homogenates containing Emx1-expressing neurons. This approach allowed us to couple translatomic with epigenomic sequencing data to evaluate the influence of histone modifications H4K8ac and H3K27me3 on translating mRNA after ELE. A subset of ELE mice underwent a hippocampal learning task to determine the gene expression and epigenetic underpinnings of ELE’s contribution to improved hippocampal memory performance. From this experiment, we discover gene expression – histone modification relationships that may play a critical role in facilitated memory after ELE. Our data reveal candidate gene-histone modification interactions and implicate gene regulatory pathways involved in ELE’s impact on hippocampal memory.