RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

Abstract Background : Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the function of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. Results : RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) neurons and to bulk VLPO samples. Gene set analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. Conclusion : Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus (VLPO) during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair, suggesting these could be driven by neuronal activity. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

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RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

10.21203/rs.2.21230/v3 ◽

2020 ◽

Author(s):

Xiaofeng Guo ◽

Xiaoling Gao ◽

Brendan T. Keenan ◽

Jingxu Zhu ◽

Dimitra Sarantopoulou ◽

...

Keyword(s):

Dna Repair ◽

Transcriptional Regulation ◽

Er Stress ◽

Differentially Expressed Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Differentially Expressed ◽

Rna Seq ◽

Preoptic Nucleus ◽

Ventrolateral Preoptic Nucleus

Abstract Background: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. Results: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. Conclusion: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

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RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

10.21203/rs.2.21230/v4 ◽

2020 ◽

Author(s):

Xiaofeng Guo ◽

Xiaoling Gao ◽

Brendan T. Keenan ◽

Jingxu Zhu ◽

Dimitra Sarantopoulou ◽

...

Keyword(s):

Dna Repair ◽

Transcriptional Regulation ◽

Er Stress ◽

Differentially Expressed Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Differentially Expressed ◽

Rna Seq ◽

Preoptic Nucleus ◽

Ventrolateral Preoptic Nucleus

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RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

BMC Genomics ◽

10.1186/s12864-020-07050-7 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Xiaofeng Guo ◽

Xiaoling Gao ◽

Brendan T. Keenan ◽

Jingxu Zhu ◽

Dimitra Sarantopoulou ◽

...

Keyword(s):

Dna Repair ◽

Transcriptional Regulation ◽

Er Stress ◽

Differentially Expressed Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Differentially Expressed ◽

Rna Seq ◽

Preoptic Nucleus ◽

Ventrolateral Preoptic Nucleus

Abstract Background Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. Results RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(−) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. Conclusion Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

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RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake

10.21203/rs.2.21230/v2 ◽

2020 ◽

Author(s):

Xiaofeng Guo ◽

Xiaoling Gao ◽

Brendan T. Keenan ◽

Jingxu Zhu ◽

Dimitra Sarantopoulou ◽

...

Keyword(s):

Dna Repair ◽

Transcriptional Regulation ◽

Er Stress ◽

Differentially Expressed Genes ◽

Rna Binding ◽

Rna Binding Proteins ◽

Differentially Expressed ◽

Rna Seq ◽

Preoptic Nucleus ◽

Ventrolateral Preoptic Nucleus

Abstract Background: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. Results: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) neurons and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. Conclusion: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

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Identification of Novel RNA Binding Proteins Influencing Circular RNA Expression in Hepatocellular Carcinoma

International Journal of Molecular Sciences ◽

10.3390/ijms22147477 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7477

Author(s):

Rok Razpotnik ◽

Petra Nassib ◽

Tanja Kunej ◽

Damjana Rozman ◽

Tadeja Režen

Keyword(s):

Hepatocellular Carcinoma ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Protein ◽

Circular Rna ◽

Differentially Expressed ◽

Circular Rnas ◽

Bioinformatics Analyses ◽

Published Evidence

Circular RNAs (circRNAs) are increasingly recognized as having a role in cancer development. Their expression is modified in numerous cancers, including hepatocellular carcinoma (HCC); however, little is known about the mechanisms of their regulation. The aim of this study was to identify regulators of circRNAome expression in HCC. Using publicly available datasets, we identified RNA binding proteins (RBPs) with enriched motifs around the splice sites of differentially expressed circRNAs in HCC. We confirmed the binding of some of the candidate RBPs using ChIP-seq and eCLIP datasets in the ENCODE database. Several of the identified RBPs were found to be differentially expressed in HCC and/or correlated with the overall survival of HCC patients. According to our bioinformatics analyses and published evidence, we propose that NONO, PCPB2, PCPB1, ESRP2, and HNRNPK are candidate regulators of circRNA expression in HCC. We confirmed that the knocking down the epithelial splicing regulatory protein 2 (ESRP2), known to be involved in the maintenance of the adult liver phenotype, significantly changed the expression of candidate circRNAs in a model HCC cell line. By understanding the systemic changes in transcriptome splicing, we can identify new proteins involved in the molecular pathways leading to HCC development and progression.

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dSreg: a Bayesian model to integrate changes in splicing and RNA-binding protein activity

Bioinformatics ◽

10.1093/bioinformatics/btz915 ◽

2019 ◽

Vol 36 (7) ◽

pp. 2134-2141

Author(s):

Carlos Martí-Gómez ◽

Enrique Lara-Pezzi ◽

Fátima Sánchez-Cabo

Keyword(s):

Bayesian Model ◽

Binding Proteins ◽

Rna Binding ◽

Environmental Changes ◽

Rna Binding Proteins ◽

Gene Set Enrichment Analysis ◽

Supplementary Information ◽

Rna Seq ◽

Protein Activity ◽

Enrichment Methods

Abstract Motivation Alternative splicing (AS) is an important mechanism in the generation of transcript diversity across mammals. AS patterns are dynamically regulated during development and in response to environmental changes. Defects or perturbations in its regulation may lead to cancer or neurological disorders, among other pathological conditions. The regulatory mechanisms controlling AS in a given biological context are typically inferred using a two-step framework: differential AS analysis followed by enrichment methods. These strategies require setting rather arbitrary thresholds and are prone to error propagation along the analysis. Results To overcome these limitations, we propose dSreg, a Bayesian model that integrates RNA-seq with data from regulatory features, e.g. binding sites of RNA-binding proteins. dSreg identifies the key underlying regulators controlling AS changes and quantifies their activity while simultaneously estimating the changes in exon inclusion rates. dSreg increased both the sensitivity and the specificity of the identified AS changes in simulated data, even at low read coverage. dSreg also showed improved performance when analyzing a collection of knock-down RNA-binding proteins’ experiments from ENCODE, as opposed to traditional enrichment methods, such as over-representation analysis and gene set enrichment analysis. dSreg opens the possibility to integrate a large amount of readily available RNA-seq datasets at low coverage for AS analysis and allows more cost-effective RNA-seq experiments. Availability and implementation dSreg was implemented in python using stan and is freely available to the community at https://bitbucket.org/cmartiga/dsreg. Supplementary information Supplementary data are available at Bioinformatics online.

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First Identification of RNA-Binding Proteins That Regulate Alternative Exons in the Dystrophin Gene

International Journal of Molecular Sciences ◽

10.3390/ijms21207803 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7803

Author(s):

Julie Miro ◽

Anne-Laure Bougé ◽

Eva Murauer ◽

Emmanuelle Beyne ◽

Dylan Da Cunha ◽

...

Keyword(s):

Large Scale ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dystrophin Gene ◽

Rna Seq ◽

Spatio Temporal ◽

Dmd Gene ◽

Shed Light

The Duchenne muscular dystrophy (DMD) gene has a complex expression pattern regulated by multiple tissue-specific promoters and by alternative splicing (AS) of the resulting transcripts. Here, we used an RNAi-based approach coupled with DMD-targeted RNA-seq to identify RNA-binding proteins (RBPs) that regulate splicing of its skeletal muscle isoform (Dp427m) in a human muscular cell line. A total of 16 RBPs comprising the major regulators of muscle-specific splicing events were tested. We show that distinct combinations of RBPs maintain the correct inclusion in the Dp427m of exons that undergo spatio-temporal AS in other dystrophin isoforms. In particular, our findings revealed the complex networks of RBPs contributing to the splicing of the two short DMD exons 71 and 78, the inclusion of exon 78 in the adult Dp427m isoform being crucial for muscle function. Among the RBPs tested, QKI and DDX5/DDX17 proteins are important determinants of DMD exon inclusion. This is the first large-scale study to determine which RBP proteins act on the physiological splicing of the DMD gene. Our data shed light on molecular mechanisms contributing to the expression of the different dystrophin isoforms, which could be influenced by a change in the function or expression level of the identified RBPs.

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Translational Control of Cytochrome c by RNA-Binding Proteins TIA-1 and HuR

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.3295-3307.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 3295-3307 ◽

Cited By ~ 136

Author(s):

Tomoko Kawai ◽

Ashish Lal ◽

Xiaoling Yang ◽

Stefanie Galban ◽

Krystyna Mazan-Mamczarz ◽

...

Keyword(s):

Er Stress ◽

Translational Control ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Patterns ◽

Mrna Levels ◽

Coding Region ◽

Translation Rate ◽

Translational Repressor

ABSTRACT Stresses affecting the endoplasmic reticulum (ER) globally modulate gene expression patterns by altering posttranscriptional processes such as translation. Here, we use tunicamycin (Tn) to investigate ER stress-triggered changes in the translation of cytochrome c, a pivotal regulator of apoptosis. We identified two RNA-binding proteins that associate with its ∼900-bp-long, adenine- and uridine-rich 3′ untranslated region (UTR): HuR, which displayed affinity for several regions of the cytochrome c 3′UTR, and T-cell-restricted intracellular antigen 1 (TIA-1), which preferentially bound the segment proximal to the coding region. HuR did not appear to influence the cytochrome c mRNA levels but instead promoted cytochrome c translation, as HuR silencing greatly diminished the levels of nascent cytochrome c protein. By contrast, TIA-1 functioned as a translational repressor of cytochrome c, with interventions to silence TIA-1 dramatically increasing cytochrome c translation. Following treatment with Tn, HuR binding to cytochrome c mRNA decreased, and both the presence of cytochrome c mRNA within actively translating polysomes and the rate of cytochrome c translation declined. Taken together, our data suggest that the translation rate of cytochrome c is determined by the opposing influences of HuR and TIA-1 upon the cytochrome c mRNA. Under unstressed conditions, cytochrome c mRNA is actively translated, but in response to ER stress agents, both HuR and TIA-1 contribute to lowering its biosynthesis rate. We propose that HuR and TIA-1 function coordinately to maintain precise levels of cytochrome c production under unstimulated conditions and to modify cytochrome c translation when damaged cells are faced with molecular decisions to follow a prosurvival or a prodeath path.

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RNA-seq reveals the RNA binding proteins, Hfq and RsmA, play various roles in virulence, antibiotic production and genomic flux in Serratia sp. ATCC 39006

BMC Genomics ◽

10.1186/1471-2164-14-822 ◽

2013 ◽

Vol 14 (1) ◽

pp. 822 ◽

Cited By ~ 21

Author(s):

Nabil M Wilf ◽

Adam J Reid ◽

Joshua P Ramsay ◽

Neil R Williamson ◽

Nicholas J Croucher ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Antibiotic Production ◽

Rna Seq

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RBPMetaDB: A comprehensive annotation of mouse RNA-Seq datasets with perturbations of RNA-binding proteins

10.1101/326116 ◽

2018 ◽

Author(s):

Jin Li ◽

Su-Ping Deng ◽

Jacob Vieira ◽

James Thomas ◽

Valerio Costa ◽

...

Keyword(s):

Gene Regulation ◽

Dna Binding ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Experimental Studies ◽

Easy Access ◽

Rna Seq ◽

Mrna Stabilization

AbstractRNA-binding proteins may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing, and mRNA stabilization via binding activities to RNA molecules. Due to the importance of RNA-binding proteins in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RNA-binding proteins were collected and integrated into a database called RBPMetaDB. This database contains 278 mouse RNA-Seq datasets for a comprehensive list of 163 RNA-binding proteins. These RNA-binding proteins account for only ∼10% of all known RNA-binding proteins annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RNA-binding proteins for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RNA-binding proteins in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RNA-binding proteins.Database URL: http://rbpmetadb.yubiolab.org

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