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The synthetic opioid fentanyl is a major contributor to the current opioid addiction crisis. We report that claustral neurons projecting to the frontal cortex limit oral fentanyl self-administration in mice. We found that fentanyl transcriptionally activates frontal-projecting claustrum neurons. These neurons also exhibit a unique suppression of Ca 2+ activity upon initiation of bouts of fentanyl consumption. Optogenetic stimulation of frontal-projecting claustral neurons, intervening in this suppression, decreased bouts of fentanyl consumption. In contrast, constitutive inhibition of frontal-projecting claustral neurons in the context of a novel, group-housed self-administration procedure increased fentanyl bout consumption. This same manipulation also sensitized conditioned-place preference for fentanyl and enhanced the representation of fentanyl experience in the frontal cortex. Together, our results indicate that claustrum neurons exert inhibitory control over frontal cortical neurons to restrict oral fentanyl intake. Upregulation of activity in the claustro-frontal projection may be a promising strategy for reducing human opioid addiction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Schmal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Here we describe a new integrative approach for accurate annotation and quantification of circRNAs named Short Read circRNA Pipeline (SRCP). Our strategy involves two steps: annotation of validated circRNAs followed by a quantification step. We show that SRCP is more sensitive than other individual pipelines and allows for more comprehensive quantification of a larger number of differentially expressed circRNAs. To facilitate the use of SRCP, we generate a comprehensive collection of validated circRNAs in five different organisms, including humans. We then utilize our approach and identify a subset of circRNAs bound to the miRNA-effector protein AGO2 in human brain samples., (© 2021. The Author(s).)

Drug addiction develops due to brain-wide plasticity within neuronal ensembles, mediated by dynamic gene expression. Though the most common approach to identify such ensembles relies on immediate early gene expression, little is known of how the activity of these genes is linked to modified behavior observed following repeated drug exposure. To address this gap, we present a broad-to-specific approach, beginning with a comprehensive investigation of brain-wide cocaine-driven gene expression, through the description of dynamic spatial patterns of gene induction in subregions of the striatum, and finally address functionality of region-specific gene induction in the development of cocaine preference. Our findings reveal differential cell-type specific dynamic transcriptional recruitment patterns within two subdomains of the dorsal striatum following repeated cocaine exposure. Furthermore, we demonstrate that induction of the IEG Egr2 in the ventrolateral striatum, as well as the cells within which it is expressed, are required for the development of cocaine seeking., Competing Interests: DM, BG, RA, HT, MG, AC No competing interests declared, (© 2021, Mukherjee et al.)

The induction of immediate-early gene (IEG) expression in brain nuclei in response to an experience is necessary for the formation of long-term memories. Additionally, the rapid dynamics of IEG induction and decay motivates the common use of IEG expression as markers for identification of neuronal assemblies ("ensembles") encoding recent experience. However, major gaps remain in understanding the rules governing the distribution of IEGs within neuronal assemblies. Thus, the extent of correlation between coexpressed IEGs, the cell specificity of IEG expression, and the spatial distribution of IEG expression have not been comprehensively studied. To address these gaps, we utilized quantitative multiplexed single-molecule fluorescence in situ hybridization (smFISH) and measured the expression of IEGs ( Arc , Egr2 , and Nr4a1 ) within spiny projection neurons (SPNs) in the dorsal striatum of mice following acute exposure to cocaine. Exploring the relevance of our observations to other brain structures and stimuli, we also analyzed data from a study of single-cell RNA sequencing of mouse cortical neurons. We found that while IEG expression is graded, the expression of multiple IEGs is tightly correlated at the level of individual neurons. Interestingly, we observed that region-specific rules govern the induction of IEGs in SPN subtypes within striatal subdomains. We further observed that IEG-expressing assemblies form spatially defined clusters within which the extent of IEG expression correlates with cluster size. Together, our results suggest the existence of IEG-expressing neuronal "superensembles," which are associated in spatial clusters and characterized by coherent and robust expression of multiple IEGs., Competing Interests: The authors declare no competing interest.

The claustrum is a small nucleus, exhibiting vast reciprocal connectivity with cortical, subcortical, and midbrain regions. Recent studies, including ours, implicate the claustrum in salience detection and attention. In the current study, we develop an iterative functional investigation of the claustrum, guided by quantitative spatial transcriptional analysis. Using this approach, we identify a circuit involving dopamine-receptor expressing claustral neurons projecting to frontal cortex necessary for context association of reward. We describe the recruitment of claustral neurons by cocaine and their role in drug sensitization. In order to characterize the circuit within which these neurons are embedded, we apply chemo- and opto-genetic manipulation of increasingly specified claustral subpopulations. This strategy resolves the role of a defined network of claustrum neurons expressing dopamine D1 receptors and projecting to frontal cortex in the acquisition of cocaine conditioned-place preference and real-time optogenetic conditioned-place preference. In sum, our results suggest a role for a claustrum-to-frontal cortex circuit in the attribution of incentive salience, allocating attention to reward-related contextual cues., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exonic circular RNAs (circRNAs) are highly abundant RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in Drosophila . There were no effects on the levels of their linear counterparts or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cellular levels and generated for the first time flies in which specific circRNAs are downregulated., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Background: The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season. An easy and convenient diagnostic tool is currently missing., Methods: To establish a validated test, we followed a 3-stage biomarker development strategy: (a) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; and these biomarkers (b) were migrated to a clinically relevant gene expression profiling platform (NanoString) and (c) were externally validated using an independent study with 28 early or late chronotypes., Results: We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard method, dim-light melatonin onset, at smaller monetary, time, and sample-number cost., Conclusion: The BodyTime assay provides a new diagnostic tool for personalization of health care according to the patient's circadian clock., Funding: This study was supported by the Bundesministerium für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.

In Drosophila, A-to-I editing is prevalent in the brain, and mutations in the editing enzyme ADAR correlate with specific behavioral defects. Here we demonstrate a role for ADAR in behavioral temperature adaptation in Drosophila. Although there is a higher level of editing at lower temperatures, at 29°C more sites are edited. These sites are less evolutionarily conserved, more disperse, less likely to be involved in secondary structures, and more likely to be located in exons. Interestingly, hypomorph mutants for ADAR display a weaker transcriptional response to temperature changes than wild-type flies and a highly abnormal behavioral response upon temperature increase. In sum, our data shows that ADAR is essential for proper temperature adaptation, a key behavior trait that is essential for survival of flies in the wild. Moreover, our results suggest a more general role of ADAR in regulating RNA secondary structures in vivo.

Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

We established two human embryonic stem cell (hESC) lines with a GGGGCC expansion in the C9orf72 gene (C9), and compared them with haploidentical and unrelated C9 induced pluripotent stem cells (iPSCs). We found a marked difference in C9 methylation between the cells. hESCs and parental fibroblasts are entirely unmethylated while the iPSCs are hypermethylated. In addition, we show that the expansion alters promoter usage and interferes with the proper splicing of intron 1, eventually leading to the accumulation of repeat-containing mRNA following neural differentiation. These changes are attenuated in C9 iPSCs, presumably owing to hypermethylation. Altogether, this study highlights the importance of neural differentiation in the pathogenesis of disease and points to the potential role of hypermethylation as a neuroprotective mechanism against pathogenic mRNAs, envisaging a milder phenotype in C9 iPSCs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) are widely expressed noncoding RNAs. However, their biogenesis and possible functions are poorly understood. Here, by studying circRNAs that we identified in neuronal tissues, we provide evidence that animal circRNAs are generated cotranscriptionally and that their production rate is mainly determined by intronic sequences. We demonstrate that circularization and splicing compete against each other. These mechanisms are tissue specific and conserved in animals. Interestingly, we observed that the second exon of the splicing factor muscleblind (MBL/MBNL1) is circularized in flies and humans. This circRNA (circMbl) and its flanking introns contain conserved muscleblind binding sites, which are strongly and specifically bound by MBL. Modulation of MBL levels strongly affects circMbl biosynthesis, and this effect is dependent on the MBL binding sites. Together, our data suggest that circRNAs can function in gene regulation by competing with linear splicing. Furthermore, we identified muscleblind as a factor involved in circRNA biogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Adiponectin receptor 1 (AdipoR1) mediates adiponectin's pleiotropic effects in muscle and liver and plays an important role in the regulation of insulin resistance and diabetes. Here, we demonstrate a pivotal role for microRNA-221 (miR-221) and the RNA-binding protein polypyrimidine tract-binding protein (PTB) in posttranscriptional regulation of AdipoR1 during muscle differentiation and in obesity. RNA-immunoprecipitation and luciferase reporter assays illustrated that both PTB and miR-221 bind AdipoR1-3'UTR and cooperatively inhibit AdipoR1 translation. Depletion of PTB or miR-221 increased, while overexpression of these factors decreased, AdipoR1 protein synthesis in both muscle and liver cells. During myogenesis, downregulation of PTB and miR-221 robustly induced AdipoR1 translation, providing a mechanism for enhanced AdipoR1 protein expression and activation in differentiated muscle cells. In addition, since both PTB and miR-221 are upregulated in liver and muscle of genetic and dietary mouse models of obesity, this novel translational mechanism may be at least partly responsible for the reduction in AdipoR1 protein levels in obesity. These findings highlight the importance of translational control in regulating AdipoR1 protein expression and adiponectin signaling. Given that adiponectin is reduced in obesity, induction of AdipoR1 could potentially enhance adiponectin beneficial effects and ameliorate insulin resistance and diabetes.

Post-transcriptional control of gene expression has central importance during development and adulthood and in physiology in general. However, little is known about the extent of post-transcriptional control of gene expression in the brain. Most post-transcriptional regulatory effectors (e.g., miRNAs) destabilize target mRNAs by shortening their polyA tails. Hence, the fraction of a given mRNA that it is fully polyadenylated should correlate with its stability and serves as a good measure of post-transcriptional control. Here, we compared RNA-seq datasets from fly brains that were generated either from total (rRNA-depleted) or polyA-selected RNA. By doing this comparison we were able to compute a coefficient that measures the extent of post-transcriptional control for each brain-expressed mRNA. In agreement with current knowledge, we found that mRNAs encoding ribosomal proteins, metabolic enzymes, and housekeeping genes are among the transcripts with least post-transcriptional control, whereas mRNAs that are known to be highly unstable, like circadian mRNAs and mRNAs expressing synaptic proteins and proteins with neuronal functions, are under strong post-transcriptional control. Surprisingly, the latter group included many specific groups of genes relevant to brain function and behavior. In order to determine the importance of miRNAs in this regulation, we profiled miRNAs from fly brains using oligonucleotide microarrays. Surprisingly, we did not find a strong correlation between the expression levels of miRNAs in the brain and the stability of their target mRNAs; however, genes identified as highly regulated post-transcriptionally were strongly enriched for miRNA targets. This demonstrates a central role of miRNAs for modulating the levels and turnover of brain-specific mRNAs in the fly.

The initial step in microRNA (miRNA) biogenesis requires processing of the precursor miRNA (pre-miRNA) from a longer primary transcript. Many pre-miRNAs originate from introns, and both a mature miRNA and a spliced RNA can be generated from the same transcription unit. We have identified a mechanism in which RNA splicing negatively regulates the processing of pre-miRNAs that overlap exon-intron junctions. Computational analysis identified dozens of such pre-miRNAs, and experimental validation demonstrated competitive interaction between the Microprocessor complex and the splicing machinery. Tissue-specific alternative splicing regulates maturation of one such miRNA, miR-412, resulting in effects on its targets that code a protein network involved in neuronal cell death processes. This mode of regulation specifically controls maturation of splice-site-overlapping pre-miRNAs but not pre-miRNAs located completely within introns or exons of the same transcript. Our data present a biological role of alternative splicing in regulation of miRNA biogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)