DNA G-Quadruplex Is a Transcriptional Control Device That Regulates Memory.

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here, we report that G-quadruplex DNA (G4-DNA) accumulates in neurons, in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory in male C57/BL6 mice. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation.One-Sentence Summary: G4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

Aberrant splicing in Huntington's disease via disrupted TDP-43 activity accompanied by altered m6A RNA modification.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene encoding huntingtin. Prior reports have established a correlation between CAG expanded HTT and altered gene expression. However, the mechanisms leading to disruption of RNA processing in HD remain unclear. Here, our analysis of the reported HTT protein interactome identifies interactions with known RNA-binding proteins (RBPs). Total, long-read sequencing and targeted RASL-seq of RNAs from cortex and striatum of the HD mouse model R6/2 reveals increased exon skipping which is confirmed in Q150 and Q175 knock-in mice and in HD human brain. We identify the RBP TDP-43 and the N6-methyladenosine (m6A) writer protein methyltransferase 3 (METTL3) to be upstream regulators of exon skipping in HD. Along with this novel mechanistic insight, we observe decreased nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 in HD mice and human brain. In addition, TDP-43 co-localizes with HTT in human HD brain forming novel nuclear aggregate-like bodies distinct from mutant HTT inclusions or previously observed TDP-43 pathologies. Binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a novel mechanism underlying alternative splicing/unannotated exon usage in HD and highlights the critical nature of TDP-43 function across multiple neurodegenerative diseases.

Fear extinction is regulated by the activity of long noncoding RNAs at the synapse.

Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules that are involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are localized to the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq, we identified a specific set of lncRNAs that accumulate in the synaptic compartment within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these was a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single-molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3BP2 and CAPRIN1, regulates the activity-dependent trafficking and clustering of RNA granules. In addition, we found that cell-type-specific, activity-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and RNA condensates in the synaptic compartment.

Synapse-Enriched m6A-Modified Malat1 Interacts with the Novel m6A Reader, DPYSL2, and Is Required for Fear-Extinction Memory.

The RNA modification N6-methyladenosine (m6A) regulates the interaction between RNA and various RNA binding proteins within the nucleus and other subcellular compartments and has recently been shown to be involved in experience-dependent plasticity, learning, and memory. Using m6A RNA-sequencing, we have discovered a distinct population of learning-related m6A- modified RNAs at the synapse, which includes the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (Malat1). RNA immunoprecipitation and mass spectrometry revealed 12 new synapse-specific learning-induced m6A readers in the mPFC of male C57/BL6 mice, with m6A-modified Malat1 binding to a subset of these, including CYFIP2 and DPYSL2. In addition, a cell type- and synapse-specific, and state-dependent, reduction of m6A on Malat1 impairs fear-extinction memory; an effect that likely occurs through a disruption in the interaction between Malat1 and DPYSL2 and an associated decrease in dendritic spine formation. These findings highlight the critical role of m6A in regulating the functional state of RNA during the consolidation of fear-extinction memory, and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.SIGNIFICANCE STATEMENT We have discovered that learning-induced m6A-modified RNA (including the long noncoding RNA, Malat1) accumulates in the synaptic compartment. We have identified several new m6A readers that are associated with fear extinction learning and demonstrate a causal relationship between m6A-modified Malat1 and the formation of fear-extinction memory. These findings highlight the role of m6A in regulating the functional state of an RNA during memory formation and expand the repertoire of experience-dependent m6A readers in the synaptic compartment.

Using 5' 32 P-labeled Primer and Reverse Transcription to Probe RNA Structure.

RNA molecules perform numerous cellular functions necessary for cell viability, some of which can depend on the RNA's structure. Therefore, it is important to study RNA structure and RNA folding to better understand the molecular basis of these functions. RNA chemical mapping strategies to elucidate RNA structural changes involve using chemical reagents that form adducts or cleave RNA. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) measures RNA flexibility by modification of the 2' hydroxyl groups of flexible nucleotides. These RNA adducts can be detected using 32 P-labeled primers and reverse transcription (RT) followed by PAGE analysis. This strategy can reveal the base-paired regions of the RNA and provide insight into tertiary structure and solvent accessibility. This protocol provides a method to interrogate RNA structure using furoyl acylimidazole (FAI). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Reverse transcription (RT) primer labeling with 32 P radionuclide Basic Protocol 2: Characterization of RNA structure with radiolabeled primer and reverse transcription (RT).

Overview of Chemical Methods to Probe RNA Structure with Radionucleotides.

Structural features of RNA play an important role in its capability to perform various functions in biological systems. To probe structural features, chemical probes are used to conjugate or cleave RNA at solvent-accessible sites, differentiating between flexible and constrained regions. These conjugates or cleaved products are then detected using reverse transcription (RT), where enzymatic RNA-dependent DNA primer extension is abruptly halted at the conjugation site or cleavage site. Here, we provide an overview of methods to probe RNA structure in vitro using radioactively labeled DNA primers, which provide a highly sensitive method to visualize RT stop sites with gel electrophoresis. © 2023 Wiley Periodicals LLC.

Cycloaddition enabled mutational profiling of 5-vinyluridine in RNA.

We report the detection of 5-vinyluridine (5-VUrd) in RNA at single nucleotide resolution via mutational profiling. Maleimide cycloadducts with 5-VUrd in RNA cause a stop in primer extension during reverse transcription, and the full-length cDNA product from reverse transcription contains misincorporation across the cycloadduct site.

Engineering an inhibitor-resistant human CSF1R variant for microglia replacement.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.

Probing the dynamic RNA structurome and its functions.

RNA is a key regulator of almost every cellular process, and the structures adopted by RNA molecules are thought to be central to their functions. The recent fast-paced evolution of high-throughput sequencing-based RNA structure mapping methods has enabled the rapid in vivo structural interrogation of entire cellular transcriptomes. Collectively, these studies are shedding new light on the long underestimated complexity of the structural organization of the transcriptome - the RNA structurome. Moreover, recent analyses are challenging the view that the RNA structurome is a static entity by revealing how RNA molecules establish intricate networks of alternative intramolecular and intermolecular interactions and that these ensembles of RNA structures are dynamically regulated to finely tune RNA functions in living cells. This new understanding of how RNA can shape cell phenotypes has important implications for the development of RNA-targeted therapeutic strategies.

An Optimized Enzyme-Nucleobase Pair Enables In Vivo RNA Metabolic Labeling with Improved Cell-Specificity.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.

mRNAs encoding neurodevelopmental regulators have equal N6-methyladenosine stoichiometry in Drosophila neuroblasts and neurons.

N6-methyladenosine (m6A) is the most prevalent internal mRNA modification in metazoans and is particularly abundant in the central nervous system. The extent to which m6A is dynamically regulated and whether m6A contributes to cell type-specific mRNA metabolism in the nervous system, however, is largely unknown. To address these knowledge gaps, we mapped m6A and measured mRNA decay in neural progenitors (neuroblasts) and neurons of the Drosophila melanogaster larval brain. We identified 867 m6A targets; 233 of these are novel and preferentially encode regulators of neuroblast proliferation, cell fate-specification and synaptogenesis. Comparison of the neuroblast and neuron m6A transcriptomes revealed that m6A stoichiometry is largely uniform; we did not find evidence of neuroblast-specific or neuron-specific m6A modification. While m6A stoichiometry is constant, m6A targets are significantly less stable in neuroblasts than in neurons, potentially due to m6A-independent stabilization in neurons. We used in vivo quantitative imaging of m6A target proteins in Mettl3 methyltransferase null brains and Ythdf m6A reader overexpressing brains to assay metabolic effects of m6A. Target protein levels decreased in Mettl3 null brains and increased in Ythdf overexpressing brains, supporting a previously proposed model in which m6A enhances translation of target mRNAs. We conclude that m6A does not directly regulate mRNA stability during Drosophila neurogenesis but is rather deposited on neurodevelopmental transcripts that have intrinsic low stability in order to augment protein output.

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

A Fluorescent Reverse-Transcription Assay to Detect Chemical Adducts on RNA.

Herein, we detail a novel reverse-transcription (RT) assay to directly detect chemical adducts on RNA. We optimize a fluorescence quenching assay to detect RT polymerization and employ our approach to detect N1-alkylation of inosine, an important post-transcriptional modification, using a phenylacrylamide as a model compound. We anticipate our approach can be expanded to identify novel reagents that form adducts with RNA and further explored to understand the relationship between RT processivity and natural post-transcriptional modifications in RNA.

Repurposing a DNA-Repair Enzyme for Targeted Protein Degradation.

Herein we present the exploration of the utility of DNA demethylase enzymes for targeted protein degradation. Novel benzylguanine substrates are characterized for their ability to control protein degradation in cells. Our data demonstrate the utility of this approach to degrade fusion proteins in different localizations within living cells.

Halo-seq: An RNA Proximity Labeling Method for the Isolation and Analysis of Subcellular RNA Populations.

The subcellular localization of specific RNA molecules promotes localized cellular activity across a variety of species and cell types. The misregulation of this RNA targeting can result in developmental defects, and mutations in proteins that regulate this process are associated with multiple diseases. For the vast majority of localized RNAs, however, the mechanisms that underlie their subcellular targeting are unknown, partly due to the difficulty associated with profiling and quantifying subcellular RNA populations. To address this challenge, we developed Halo-seq, a proximity labeling technique that can label and profile local RNA content at virtually any subcellular location. Halo-seq relies on a HaloTag fusion protein localized to a subcellular space of interest. Through the use of a radical-producing Halo ligand, RNAs that are near the HaloTag fusion are specifically labeled with spatial and temporal control. Labeled RNA is then specifically biotinylated in vitro via a click reaction, facilitating its purification from a bulk RNA sample using streptavidin beads. The content of the biotinylated RNA is then profiled using high-throughput sequencing. In this article, we describe the experimental and computational procedures for Halo-seq, including important benchmark and quality control steps. By allowing the flexible profiling of a variety of subcellular RNA populations, we envision Halo-seq facilitating the discovery and further study of RNA localization regulatory mechanisms. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualization of HaloTag fusion protein localization Basic Protocol 2: In situ copper-catalyzed cycloaddition of fluorophore via click reaction Basic Protocol 3: In vivo RNA alkynylation and extraction of total RNA Basic Protocol 4: In vitro copper-catalyzed cycloaddition of biotin via click reaction Basic Protocol 5: Assessment of RNA biotinylation by RNA dot blot Basic Protocol 6: Enrichment of biotinylated RNA using streptavidin beads and preparation of RNA-seq library Basic Protocol 7: Computational analysis of Halo-seq data.

An atlas of posttranslational modifications on RNA binding proteins.

RNA structure and function are intimately tied to RNA binding protein recognition and regulation. Posttranslational modifications are chemical modifications which can control protein biology. The role of PTMs in the regulation RBPs is not well understood, in part due to a lacking analysis of PTM deposition on RBPs. Herein, we present an analysis of posttranslational modifications (PTMs) on RNA binding proteins (RBPs; a PTM RBP Atlas). We curate published datasets and primary literature to understand the landscape of PTMs and use protein-protein interaction data to understand and potentially provide a framework for understanding which enzymes are controlling PTM deposition and removal on the RBP landscape. Intersection of our data with The Cancer Genome Atlas also provides researchers understanding of mutations that would alter PTM deposition. Additional characterization of the RNA-protein interface provided from in-cell UV crosslinking experiments provides a framework for hypotheses about which PTMs could be regulating RNA binding and thus RBP function. Finally, we provide an online database for our data that is easy to use for the community. It is our hope our efforts will provide researchers will an invaluable tool to test the function of PTMs controlling RBP function and thus RNA biology.

Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo.

Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.

ADRAM is an experience-dependent long noncoding RNA that drives fear extinction through a direct interaction with the chaperone protein 14-3-3.

Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.

The P522R protective variant of PLCG2 promotes the expression of antigen presentation genes by human microglia in an Alzheimer's disease mouse model.

The P522R variant of PLCG2, expressed by microglia, is associated with reduced risk of Alzheimer's disease (AD). Yet, the impact of this protective mutation on microglial responses to AD pathology remains unknown. Chimeric AD and wild-type mice were generated by transplanting PLCG2-P522R or isogenic wild-type human induced pluripotent stem cell microglia. At 7 months of age, single-cell and bulk RNA sequencing, and histological analyses were performed. The PLCG2-P522R variant induced a significant increase in microglial human leukocyte antigen (HLA) expression and the induction of antigen presentation, chemokine signaling, and T cell proliferation pathways. Examination of immune-intact AD mice further demonstrated that the PLCG2-P522R variant promotes the recruitment of CD8+ T cells to the brain. These data provide the first evidence that the PLCG2-P522R variant increases the capacity of microglia to recruit T cells and present antigens, promoting a microglial transcriptional state that has recently been shown to be reduced in AD patient brains.