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Darnell, R. B.

Publications and source records attributed to Darnell, R. B..

15 recordsLinked to original sources

Local translation couples synaptic activity to mitochondrial adaptation in dendrites

Neurons rely on localized protein synthesis to rapidly adapt synaptic function to activity, yet how dendritic translation regulates mitochondrial remodeling during synaptic plasticity remains poorly understood. Here, we show that neuronal activity engages a spatially restricted translational program that couples local protein synthesis to mitochondrial function through the non-canonical translation initiation factor eIF4G2. Using proximity labeling to profile the dendritic RNA interactome, translatome, and proteome, we identify a cohort of nuclear-encoded mitochondrial mRNAs that are selectively recruited for translation following depolarization and mGluR activation. This program drives activity-dependent increases in mitochondrial membrane potential, mitochondrial abundance, and oxygen consumption. Loss of eIF4G2 abolishes these responses, whereas dendrite-specific, but not soma-restricted, rescue restores mitochondrial remodeling, demonstrating that eIF4G2 functions locally at postsynaptic sites. Mechanistically, eIF4G2 binds the 5 prime or minute untranslated regions of activity-responsive mitochondrial transcripts and promotes translation of both upstream open reading frames (uORFs) and downstream coding sequences. Using a dendritically targeted split-GFP reporter, we further show that neuronal activity induces local uORF translation to generate previously unannotated micropeptides. Together, our findings identify eIF4G2-dependent local translation as a mechanism that establishes mitochondrial competence during synaptic activity by coordinating the production of mitochondrial proteins and uORF-encoded micropeptides.

neuroscience↗

Synovial transcriptional clusters link cartilage degeneration to cell-type-specific gene expression in knee osteoarthritis

ObjectivesTo identify synovial transcriptional clusters in human knee osteoarthritis (OA) and determine how these relate to synovial histologic features, cell-type-associated gene expression, and cartilage degeneration severity. MethodsBulk RNA sequencing (RNA-seq) of synovial tissue from n = 135 patients with knee OA was analyzed using consensus clustering. Clusters were compared by clinical and histologic features, including cartilage degeneration severity (OARSI score). Single-cell RNA-seq (n = 18) and spatial transcriptomics were used to relate cartilage degeneration-associated gene expression patterns to synovial cell populations. ResultsFour synovial transcriptional clusters that differed in synovial histologic features and cartilage degeneration severity were identified. Greater cartilage degeneration was associated with enrichment of lining fibroblast- and inflammatory myeloid-associated gene expression, whereas lesser cartilage degeneration was associated with enrichment of sublining fibroblast, endothelial, mural cell, and adipocyte-associated gene expression. ConclusionsHuman knee OA synovium segregates into transcriptional clusters associated with cartilage degeneration severity. Synovial transcriptional heterogeneity corresponds to cell-type-associated gene expression. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIOsteoarthritis synovium exhibits marked histologic and molecular heterogeneity. C_LIO_LISynovial inflammation detected by MRI correlates with cartilage degeneration and predicts progressive cartilage loss in knee OA. C_LIO_LIPrior transcriptomic studies have identified molecular subsets of OA synovium, but their relationship to cartilage degeneration severity remains unclear. C_LI What this study addsO_LIOA synovium segregates into four transcriptional clusters: Sublining (C1), Lymphomyeloid (C2), Myeloid (C3), and Major trauma (C4). C_LIO_LIGreater cartilage degeneration is associated with enrichment of inflammatory myeloid and lining fibroblast gene expression, whereas lesser degeneration is associated with enrichment of adipocyte, sublining fibroblast, endothelial, and mural cell-associated gene expression. C_LI How this study might affect research, practice or policyO_LIProvides a framework for a clinically relevant biological stratification of OA patients based on synovial molecular features. C_LIO_LIInforms future efforts to link synovial biology with OA prognosis, cartilage degeneration, treatment allocation, and development of targeted therapeutic strategies. C_LI

genomics↗

Aging-dependent dysregulation of EXOSC2 is maintained in cancer as a dependency

Reprogramming of aged donor tissue cells into induced pluripotent stem cells (A-iPSC) preserved the epigenetic memory of aged-donor tissue, defined as genomic instability and poor tissue differentiation in our previous study. The unbalanced expression of RNA exosome subunits affects the RNA degradation complex function and is associated with geriatric diseases including premature aging and cancer progression. We hypothesized that the age-dependent progressive subtle dysregulation of EXOSC2 (exosome component 2) causes the aging traits (abnormal cell cycle and poor tissue differentiation). We used embryonic stem cells as a tool to study EXOSC2 function as the aging trait epigenetic memory determined in A-iPSC because these aging traits could not be studied in senesced aged cells or immortalized cancer cells. We found that the regulatory subunit of PP2A phosphatase, PPP2R5E, is a key target of EXOSC2 and this regulation is preserved in stem cells and cancer.

cell biology↗

ELAVL3 regulates splicing of RNAs encoding synaptic signaling proteins in D1 and D2 striatal medium spiny neurons.

The neuronal RNA-binding protein (RBP) family nELAVL regulates key neuronal processes by binding directly to target RNA transcripts. In this study, we demonstrate that ELAVL3 is the predominant nELAVL paralog expressed in D1 and D2 medium spiny neurons of the striatum. To investigate its function, we developed ELAVL3 cTag-crosslinking and immunoprecipitation (CLIP) to generate RBP-RNA interaction maps from these neurons. By integrating data from ELAVL3-cTag and Elavl3 knockout mice, we identified distinct regulatory effects of ELAVL3 on alternative splicing of its target transcripts. Notably, ELAVL3 modulates splicing of transcripts encoding proteins critical for glutamate and dopamine receptor signaling. These findings underscore the role of ELAVL3 in RNA-mediated regulation of molecular pathways essential for medium spiny neuron function in the striatum.

neuroscience↗

Opto-CLIP reveals dynamic FMRP regulation of mRNAs upon CA1 neuronal activation

Neuronal diversity and function are intricately linked to the dynamic regulation of RNA metabolism. Electrophysiologic studies of synaptic plasticity, models for learning and memory, are disrupted in Fragile X Syndrome (FXS). FXS is characterized by the loss of FMRP, an RNA-binding protein (RBP) known to suppress translation of specific neuronal RNAs. Synaptic plasticity in CA1 excitatory hippocampal neurons is protein-synthesis dependent, suggesting a role for FMRP in FXS-related synaptic deficits. To explore this model, we developed Opto-CLIP, integrating optogenetics with cell-type specific FMRP-CLIP and RiboTag in CA1 neurons, allowing investigation of activity-induced FMRP regulation. We tracked changes in FMRP binding and ribosome-associated RNA profiles 30 minutes after neuronal activation. Our findings reveal distinct temporal dynamics for FMRP transcript regulation in the cell body versus the synapse. In the cell body, FMRP binding to transcripts encoding nuclear functions is relieved, potentially allowing rapid transcriptional responses to neuronal activation. At the synapse, FMRP binding to transcripts encoding synaptic targets was relatively stable, with variability in translational control across target categories. These results offer fresh insights into the dynamic regulation of RNA by FMRP in response to neuronal activation and provide a foundation for future research into the mechanisms of RBP-mediated synaptic plasticity.

neuroscience↗

MicroRNA-218 instructs proper assembly of hippocampal networks

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. A key aspect of this developmental program is mediated at the post-transcriptional level by microRNAs (miRNAs). Deletion of neuronal enriched miRNAs induces strong developmental phenotypes, and multiple reports have found altered levels of miRNAs in patients with neurodevelopmental disorders. However, cellular and molecular mechanisms used by miRNAs to instruct proper brain development remain largely unexplored. Here, through multiple screens, we identified miR-218 as a critical regulator of hippocampal assembly in mice. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons and GABAergic inhibitory interneurons. Transient inhibition of miR-218 in early life results in an adult brain with heightened network activity and a predisposition to seizures. We used RNA-seq and FACS-seq (fluorescence-activated cell sorting followed by RNA-seq) to identify global and cell type-specific changes in gene expression in the absence of miR-218 and narrow down which altered developmental processes would lead to long-term network instability. We find that miR-218 inhibition results in the disruption of early depolarizing GABAergic signaling, structural defects in dendritic spines, and altered intrinsic membrane excitability. Finally, conditional knockout of miR-218 in interneurons, but not pyramidal neurons is sufficient to recapitulate the effects on long-term stability. Taken together, the data suggest that miR-218 orchestrates hippocampal network assembly to produce a stable network in the adult, primarily by regulating interneuron function in early postnatal life.

neuroscience↗

NOVA2 regulates neural circRNA biogenesis

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establish a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circ-Efnb2 was impaired when YCAY clusters located in flanking introns were removed. CLIP and additional reporter analysis demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain.

genomics↗

AGO HITS-CLIP in Adipose Tissue Reveals miR-29 as a Post-Transcriptional Regulator of Leptin

MicroRNAs (miRNAs) are short, non-coding RNAs that associate with Argonaute (AGO) to regulate mRNA stability and translation. While individual miRNAs have been shown to play important roles in white and brown adipose tissue in normal physiology and disease1,2,3, a comprehensive analysis of miRNA activity in these tissues has not been performed. We used high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to comprehensively characterize the network of high-confidence, in vivo mRNA:miRNA interactions across white and brown fat, revealing over 100,000 unique miRNA binding sites. Targets for each miRNA were ranked to generate a catalog of miRNA binding activity, and the miR-29 family emerged as a top regulator of adipose tissue gene expression. Among the top targets of miR-29 was leptin, an adipocyte-derived hormone that acts on the brain to regulate food intake and energy expenditure4. Two independent miR-29 binding sites in the leptin 3-UTR were validated using luciferase assays, and miR-29 gain and loss-of-function modulated leptin mRNA and protein secretion in primary adipocytes. In mice, miR-29 abundance inversely correlated with leptin levels in two independent models of obesity. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies the first known post-transcriptional regulator of leptin. Future work aimed at manipulating miR-29:leptin binding may provide a therapeutic opportunity to treat obesity and its sequelae.

cell biology↗

FMRP binding to a ranked subset of long genes is revealed by coupled CLIP and TRAP in specific neuronal cell types

Loss of function of the Fragile X Mental Retardation Protein (FMRP) in human Fragile X Syndrome (FXS) and in model organisms results in phenotypes of abnormal neuronal structure and dynamics, synaptic function and connectivity which may contribute to a state of neuronal, circuit and organism hyperexcitability. Previous in vivo identification of FMRP association with specific mRNA targets in mouse brain revealed that FMRP regulates the translation of a large fraction of the synaptic proteome in both pre- and post-synaptic compartments as well as many transcription factors and chromatin modifying proteins. However, it was not previously possible to determine the ratio of FMRP binding to transcript abundance due to the complexity of different neuronal cell types in whole brain. Moreover, it has been difficult to link the translational regulation of specific targets to model phenotypes or human symptoms. For example, loss-of-function of FMRP in the Purkinje cells of the cerebellum results in three cell autonomous phenotypes related to learning and memory, including enhanced mGluR-LTD at parallel fiber synapses, altered dendritic spines and behavioral deficits in a eyeblink-conditioning learning paradigm shared by human FXS patients. The molecular basis for these and related human Fragile X phenotypes is unknown. To address these critical issues we have developed a new mouse model (the Fmr1 cTAG mouse) in which endogenous FMRP can be conditionally tagged for RNA:protein crosslinking and immunoprecipitation (CLIP) identification of the RNAs with which it interacts in vivo. We used the Fmr1 cTAG mouse to quantitatively evaluate FMRP-mRNA association in Purkinje and cerebellar granule neurons which together comprise the parallel-fiber synapse. We calculated a stoichiometrically ranked list of FMRP RNA binding events by normalizing to ribosome-associated transcript abundance determined by TRAP-seq, and now definitively find that FMRP associates with specific sets of mRNAs which differ between the two cell types. In Purkinje cells, many components of the mGluR signaling pathway are FMRP targets including the top-ranked Purkinje cell mRNA Itpr1, encoding the IP3 receptor, the function of which is critical to proper mGluR-dependent synaptic plasticity. In sum, this novel approach provides the first ranked list of FMRP target mRNAs and further reveals that FMRP regulates a specific set of long neural genes related to relevant cell autonomous phenotypes.\n\nHighlightsO_LIWe have created a mouse model in which endogenous FMRP can be conditionally tagged.\nC_LIO_LIUsing tag-specific CLIP we describe ranked and specific sets of in vivo FMRP mRNA targets in two types of neurons.\nC_LIO_LIThis ranking was used to reveal that FMRP regulates mRNAs with long coding sequences.\nC_LIO_LIFMRP mRNA targets in Purkinje cells, including the top-ranked IP3 receptor, are related to cell-autonomous Fragile X phenotypes.\nC_LIO_LIWe have updated our previous list of whole mouse brain FMRP mRNA targets with more replicates, deeper sequencing and improved analysis\nC_LIO_LIThe use of tagged FMRP in less abundant cell populations allowed identification of novel mRNA targets missed in a whole brain analysis\nC_LI

neuroscience↗

N6-methyladenosine dynamics during early vertebrate embryogenesis

Early vertebrate embryogenesis is characterized by extensive post-transcriptional regulation during the maternal-to-zygotic transition. The N6-methyladenosine (m6A) modifications on mRNA have been shown to affect both translation and stability of transcripts. Here we investigate the m6A topology during early vertebrate embryogenesis and its association with polyadenylated mRNA levels. The majority (>70%) of maternal transcripts harbor m6A, and there is a substantial increase of m6A in the polyadenylated mRNA fraction between 0 and 2 hours post fertilization. Notably, we find strong associations between m6A, cytoplasmic polyadenylation and translational efficiency prior to zygotic genome activation (ZGA). Interestingly, the relationship between m6A and translation is strongest for peaks located in the 3UTR, but not overlapping stop codons. Sequence analyses revealed enrichment of motifs for RNA binding proteins involved in translational regulation and RNA degradation. After ZGA, m6A seem to diminish the effect of miR-430 mediated degradation. The reported results improve our understanding of the combinatorial code behind post-transcriptional mRNA regulation during embryonic reprogramming and early differentiation.

developmental biology↗

Differential 3’ Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.

neuroscience↗

A master regulatory network restoring brain glutamate homeostasis is coordinately activated in stroke

Post-transcriptional regulation by miRNAs is essential for complex molecular responses to physiological insult and disease. Although many disease-associated miRNAs are known, their global targets and culminating network effects on pathophysiology remain poorly understood. We applied AGO CLIP to systematically elucidate altered miRNA-target interactions in brain following ischemia/reperfusion (I/R) injury. Among 1,190 identified, most prominent was the cumulative loss of target regulation by miR-29 family members. Integration of translational and time-course RNA profiles revealed a dynamic mode of miR-29 target de-regulation, led by acute translational activation and later increase in RNA levels, allowing rapid proteomic changes to take effect. These functional regulatory events rely on canonical and non-canonical miR-29 binding and engage glutamate reuptake signals to control local glutamate levels. These results uncover a miRNA target network that acts acutely to maintain brain homeostasis after ischemic stroke.

neuroscience↗

Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell-types in vivo is unknown. We employed NOVA2 cTag-CLIP to generate functional RBP-RNA maps from single neuronal populations in the mouse brain. Combining cell-type specific data from Nova2-cTag and Nova2 conditional knock-out mice revealed differential NOVA2 regulatory actions (e.g. alternative splicing) on the same transcripts in different neurons, including in cerebellar Purkinje cells, where NOVA2 acts as an essential factor for proper motor coordination and synapse formation. This also led to the discovery of a mechanism by which NOVA2 action leads to different outcomes in different cells on the same transcripts: NOVA2 is able to regulate retained introns, which subsequently serve as scaffolds for another trans-acting splicing factor, PTBP2. Our results describe differential roles and mechanisms by which RBPs mediate RNA diversity in different neurons and consequent functional outcomes within the brain.

neuroscience↗

De novo indels within introns contribute to ASD incidence

Copy number profiling and whole-exome sequencing has allowed us to make remarkable progress in our understanding of the genetics of autism over the past ten years, but there are major aspects of the genetics that are unresolved. Through whole-genome sequencing, additional types of genetic variants can be observed. These variants are abundant and to know which are functional is challenging. We have analyzed whole-genome sequencing data from 510 of the Simons Simplex Collections quad families and focused our attention on intronic variants. Within the introns of 546 high-quality autism target genes, we identified 63 de novo indels in the affected and only 37 in the unaffected siblings. The difference of 26 events is significantly larger than expected (p-val = 0.01) and using reasonable extrapolation shows that de novo intronic indels can contribute to at least 10% of simplex autism. The significance increases if we restrict to the half of the autism targets that are intolerant to damaging variants in the normal human population, which half we expect to be even more enriched for autism genes. For these 273 targets we observe 43 and 20 events in affected and unaffected siblings, respectively (p-value of 0.005). There was no significant signal in the number of de novo intronic indels in any of the control sets of genes analyzed. We see no signal from de novo substitutions in the introns of target genes.

genetics↗

Single-Cell RNA-Seq Of Rheumatoid Arthritis Synovial Tissue Using Low Cost Microfluidic Instrumentation

Droplet-based single cell RNA-seq has emerged as a powerful technique for massively parallel cellular profiling. While these approaches offer the exciting promise to deconvolute cellular heterogeneity in diseased tissues, the lack of cost-effective, reliable, and user-friendly instrumentation has hindered widespread adoption of droplet microfluidic techniques. To address this, we have developed a microfluidic control instrument that can be easily assembled from 3D printed parts and commercially available components costing approximately $540. We adapted this instrument for massively parallel scRNA-seq and deployed it in a clinical environment to perform single cell transcriptome profiling of disaggregated synovial tissue from a rheumatoid arthritis patient. We sequenced 8,716 single cells from a synovectomy, revealing 16 transcriptomically distinct clusters. These encompass a comprehensive and unbiased characterization of the autoimmune infiltrate, including inflammatory T and NK subsets that contribute to disease biology. Additionally, we identified fibroblast subpopulations that are demarcated via THY1 (CD90) and CD55 expression. Further experiments confirm that these represent synovial fibroblasts residing within the synovial intimal lining and subintimal lining, respectively, each under the influence of differing microenvironments. We envision that this instrument will have broad utility in basic and clinical settings, enabling low-cost and routine application of microfluidic techniques, and in particular single-cell transcriptome profiling.

genomics↗