scholarly journals An in vivo strategy for knockdown of circular RNAs

2020 ◽  
Author(s):  
Nagarjuna Reddy Pamudurti ◽  
Ines Lucia Patop ◽  
Aishwarya Krishnamoorthy ◽  
Reut Ashwal-Fluss ◽  
Osnat Bartok ◽  
...  
Keyword(s):  
Nervous System ◽  
Methodological Approach ◽  
Splice Junction ◽  
Circular Rnas ◽  
Protein Coding ◽  
Evolutionarily Conserved ◽  
Computational Platform ◽  
Target Effects ◽  
Comprehensive Study

ABSTRACTExonic circular RNAs (circRNAs) are highly abundant and evolutionarily conserved 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 miRNA-derived 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 the fly nervous system. There were no effects on levels of the linear RNA 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, we found that 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 cell levels.

scholarly journals An in vivo knockdown strategy reveals multiple functions for circMbl

2018 ◽  
Author(s):  
Nagarjuna Reddy Pamudurti ◽  
Vinay Vikas Konakondla-Jacob ◽  
Aishwarya Krishnamoorthy ◽  
Reut Ashwal-Fluss ◽  
Osnat Bartok ◽  
...  
Keyword(s):  
Gene Expression ◽  
Central Nervous System ◽  
Methodological Approach ◽  
Synaptic Function ◽  
Circular Rnas ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Target Effects ◽  
Comprehensive Study

ABSTRACTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. circRNAs are enriched in the brain and accumulate with age in flies, worms and mice. Despite their abundance, little is known about their functions, especially in the context of whole organisms. Here we report the development and use of shRNAs to knock down and study the function of circMbl, the most abundant circRNA in Drosophila. This circRNA is highly conserved through evolution and is generated from the locus of the essential splicing factor muscleblind (mbl). Briefly, we generated flies in which circMbl is reduced more than 90% without measurable off-target effects in the hosting gene as well as in other RNAs. These flies display specific defects that suggest roles of circMbl in muscle and neural tissues during development and in adult flies. More specifically, whole organism downregulation of circMbl leads to male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. Moreover, these phenotypes are recapitulated by a second shRNA targeting circMbl. Importantly, knockdown and overexpression of circMbl affect mostly the same genes but in the opposite direction. Last but not least, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level likely by acting in multiple tissues. Moreover, here we provide the first proof of functionality of circRNAs in Drosophila as well as a methodological approach that enables the comprehensive study of circRNAs in vivo.SIGNIFICANCE STATEMENTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. Here we report the development and use of a shRNA-based system to knockdown specific circRNAs in vivo. We generated flies in which circMbl, the most abundant circRNA is reduced more than 90% without measurable off-target effects. These flies display male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. These phenotypes are recapitulated by a second shRNA targeting circMbl. Moreover, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level and provide a methodological approach that enables the comprehensive study of circRNAs in vivo.

scholarly journals Circular RNAs in the Central Nervous System

2021 ◽  
Vol 8 ◽  
Author(s):  
Meng-Lan Li ◽  
Wen Wang ◽  
Zi-Bing Jin
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Circular Rnas ◽  
Protein Coding ◽  
Current State ◽  
Specific Distribution ◽  
Evolutionarily Conserved ◽  
Protein Functions ◽  
The Central Nervous System ◽  
Regulate Protein

Circular RNAs (circRNAs) are endogenous single-stranded RNAs characterized by covalently closed loop structures with neither 5′ to 3′ polarity nor poly(A) tails. They are generated most commonly from back-splicing of protein-coding exons. CircRNAs have a tissue-specific distribution and are evolutionarily conserved, and many circRNAs play important biological functions by combining with microRNAs and proteins to regulate protein functions and their own translation. Numerous studies have shown that circRNAs are enriched in the central nervous system (CNS) and play an important role in the development and maintenance of homeostasis. Correspondingly, they also play an important role in the occurrence and progression of CNS diseases. In this review, we highlight the current state of circRNA biogenesis, properties, function and the crucial roles they play in the CNS.

scholarly journals Characterization of More Selective Central Nervous System Nrf2-Activating Novel Vinyl Sulfoximine Compounds Compared to Dimethyl Fumarate

2020 ◽  
Vol 17 (3) ◽  
pp. 1142-1152 ◽  
Author(s):  
Karl E. Carlström ◽  
Praveen K. Chinthakindi ◽  
Belén Espinosa ◽  
Faiez Al Nimer ◽  
Elias S. J. Arnér ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
De Novo ◽  
Dimethyl Fumarate ◽  
The Novel ◽  
The Central Nervous System ◽  
Demyelinating Disorders ◽  
Target Effects

Abstract The Nrf2 transcription factor is a key regulator of redox reactions and considered the main target for the multiple sclerosis (MS) drug dimethyl fumarate (DMF). However, exploration of additional Nrf2-activating compounds is motivated, since DMF displays significant off-target effects and has a relatively poor penetrance to the central nervous system (CNS). We de novo synthesized eight vinyl sulfone and sulfoximine compounds (CH-1–CH-8) and evaluated their capacity to activate the transcription factors Nrf2, NFκB, and HIF1 in comparison with DMF using the pTRAF platform. The novel sulfoximine CH-3 was the most promising candidate and selected for further comparison in vivo and later an experimental model for traumatic brain injury (TBI). CH-3 and DMF displayed comparable capacity to activate Nrf2 and downstream transcripts in vitro, but with less off-target effects on HIF1 from CH-3. This was verified in cultured microglia and oligodendrocytes (OLs) and subsequently in vivo in rats. Following TBI, DMF lowered the number of leukocytes in blood and also decreased axonal degeneration. CH-3 preserved or increased the number of pre-myelinating OL. While both CH-3 and DMF activated Nrf2, CH-3 showed less off-target effects and displayed more selective OL associated effects. Further studies with Nrf2-acting compounds are promising candidates to explore potential myelin protective or regenerative effects in demyelinating disorders.

scholarly journals Closing the circle: current state and perspectives of circular RNA databases

2020 ◽  
Author(s):  
Marieke Vromman ◽  
Jo Vandesompele ◽  
Pieter-Jan Volders
Keyword(s):  
Splice Junction ◽  
Circular Rna ◽  
Circular Rnas ◽  
Working Mechanism ◽  
Comprehensive Overview ◽  
Specific Expression ◽  
Protein Coding ◽  
Rna Molecules ◽  
Current State ◽  
Coding Potential

Abstract Circular RNAs (circRNAs) are covalently closed RNA molecules that have been linked to various diseases, including cancer. However, a precise function and working mechanism are lacking for the larger majority. Following many different experimental and computational approaches to identify circRNAs, multiple circRNA databases were developed as well. Unfortunately, there are several major issues with the current circRNA databases, which substantially hamper progression in the field. First, as the overlap in content is limited, a true reference set of circRNAs is lacking. This results from the low abundance and highly specific expression of circRNAs, and varying sequencing methods, data-analysis pipelines, and circRNA detection tools. A second major issue is the use of ambiguous nomenclature. Thus, redundant or even conflicting names for circRNAs across different databases contribute to the reproducibility crisis. Third, circRNA databases, in essence, rely on the position of the circRNA back-splice junction, whereas alternative splicing could result in circRNAs with different length and sequence. To uniquely identify a circRNA molecule, the full circular sequence is required. Fourth, circRNA databases annotate circRNAs’ microRNA binding and protein-coding potential, but these annotations are generally based on presumed circRNA sequences. Finally, several databases are not regularly updated, contain incomplete data or suffer from connectivity issues. In this review, we present a comprehensive overview of the current circRNA databases and their content, features, and usability. In addition to discussing the current issues regarding circRNA databases, we come with important suggestions to streamline further research in this growing field.

scholarly journals Identification of Abundant and Evolutionarily Conserved Opioid Receptor Circular RNAs in the Nervous System Modulated by Morphine

2019 ◽  
Vol 96 (2) ◽  
pp. 247-258
Author(s):  
Takeshi Irie ◽  
Rebecca Shum ◽  
Ioanna Deni ◽  
Amanda Hunkele ◽  
Valerie Le Rouzic ◽  
...  
Keyword(s):  
Nervous System ◽  
Opioid Receptor ◽  
Circular Rnas ◽  
Evolutionarily Conserved

scholarly journals circRNAome Profiling in Oral Carcinoma Unveils a Novel circFLNB that Mediates Tumour Growth-Regulating Transcriptional Response

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1868
Author(s):  
Yi-Tung Chen ◽  
Ian Yi-Feng Chang ◽  
Chia-Hua Kan ◽  
Yu-Hao Liu ◽  
Yu-Ping Kuo ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Transcriptional Response ◽  
Transcriptome Profiling ◽  
Circular Rnas ◽  
Bioinformatics Analyses ◽  
Protein Coding ◽  
Sequencing Technologies ◽  
Oral Malignancies

Deep sequencing technologies have revealed the once uncharted non-coding transcriptome of circular RNAs (circRNAs). Despite the lack of protein-coding potential, these unorthodox yet highly stable RNA species are known to act as critical gene regulatory hubs, particularly in malignancies. However, their mechanistic implications in tumor outcome and translational potential have not been fully resolved. Using RNA-seq data, we profiled the circRNAomes of tumor specimens derived from oral squamous cell carcinoma (OSCC), which is a prevalently diagnosed cancer with a persistently low survival rate. We further catalogued dysregulated circRNAs in connection with tumorigenic progression. Using comprehensive bioinformatics analyses focused on co-expression maps and miRNA-interaction networks, we delineated the regulatory networks that are centered on circRNAs. Interestingly, we identified a tumor-associated, pro-tumorigenic circRNA, named circFLNB, that was implicated in maintaining several tumor-associated phenotypes in vitro and in vivo. Correspondingly, transcriptome profiling of circFLNB-knockdown cells showed alterations in tumor-related genes. Integrated in silico analyses further deciphered the circFLNB-targeted gene network. Together, our current study demarcates the OSCC-associated circRNAome, and unveils a novel circRNA circuit with functional implication in OSCC progression. These systems-based findings broaden mechanistic understanding of oral malignancies and raise new prospects for translational medicine.

scholarly journals The RNA landscape of the human placenta in health and disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sungsam Gong ◽  
Francesca Gaccioli ◽  
Justyna Dopierala ◽  
Ulla Sovio ◽  
Emma Cook ◽  
...  
Keyword(s):  
Small Rnas ◽  
Web Application ◽  
Large Scale ◽  
Elevated Serum ◽  
Splice Junction ◽  
Serum Levels ◽  
Circular Rnas ◽  
Protein Coding ◽  
Health And Disease ◽  
Short And Long Term

AbstractThe placenta is the interface between mother and fetus and inadequate function contributes to short and long-term ill-health. The placenta is absent from most large-scale RNA-Seq datasets. We therefore analyze long and small RNAs (~101 and 20 million reads per sample respectively) from 302 human placentas, including 94 cases of preeclampsia (PE) and 56 cases of fetal growth restriction (FGR). The placental transcriptome has the seventh lowest complexity of 50 human tissues: 271 genes account for 50% of all reads. We identify multiple circular RNAs and validate 6 of these by Sanger sequencing across the back-splice junction. Using large-scale mass spectrometry datasets, we find strong evidence of peptides produced by translation of two circular RNAs. We also identify novel piRNAs which are clustered on Chr1 and Chr14. PE and FGR are associated with multiple and overlapping differences in mRNA, lincRNA and circRNA but fewer consistent differences in small RNAs. Of the three protein coding genes differentially expressed in both PE and FGR, one encodes a secreted protein FSTL3 (follistatin-like 3). Elevated serum levels of FSTL3 in pregnant women are predictive of subsequent PE and FGR. To aid visualization of our placenta transcriptome data, we develop a web application (https://www.obgyn.cam.ac.uk/placentome/).

Monitoring stem cell migration in the nervous system by in vivo magnetic resonance imaging

2005 ◽  
Vol 25 (1_suppl) ◽  
pp. S692-S692
Author(s):  
Mathias Hoehn ◽  
Uwe Himmelreich ◽  
Ralph Weber ◽  
Pedro Ramos-Cabrer ◽  
Susanne Wegener ◽  
...  
Keyword(s):  
Magnetic Resonance Imaging ◽  
Nervous System ◽  
Stem Cell ◽  
Cell Migration ◽  
Magnetic Resonance ◽  
Resonance Imaging ◽  
Stem Cell Migration

Neuropharmacological Profile and Chemical Analysis of Moroccan Ormenis mixta L.

2018 ◽  
Vol 16 (S1) ◽  
pp. S55-S64
Author(s):  
G. Hajjaj ◽  
A. Bahlouli ◽  
M. Tajani ◽  
K. Alaoui ◽  
Y. Cherrah ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Body Weight ◽  
Traditional Medicine ◽  
Behavioral Tests ◽  
Phytochemical Screening ◽  
Activity Profile ◽  
Acute Toxicity Study ◽  
Pharmacological Studies

Ormenis mixta L. is traditionally used for central nervous system (CNS)-related diseases. Its anti-stress properties have received attention in Moroccan traditional medicine and aromatherapy. However, no pharmacological studies have yet been undertaken on this plant in Morocco. The present study provides a preliminary phytochemical screening and psychopharmacological profile of the essential oil and aqueous extract from Ormenis mixta L. by using behavioral tests in vivo, at graded doses. The result of this research shows that Ormenis mixta L. was safe up to 2 g/kg b.w. (body weight) in the acute toxicity study, possesses potential psychostimulant effect, and has antianxiety and antidepressant-like activity. This activity profile of Ormenis mixta L. was similar to the typical psychostimulant, caffeine. The exact mechanism of action underlying this stimulant-like effect should be clarified with further detailed studies. These results explained the extensive use of Ormenis mixta L. as a traditional medicine in Morocco.

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