Genetic tools for the stable overexpression of circular RNAs

Circular RNAs (circRNAs) are a class of non-coding RNAs that feature a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient overexpression of circRNAs in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs. We further showed that circRNA transposons can be supplied to mouse livers via hydrodynamic tail vein injection, resulting in ectopic circRNA expression in a hepatocellular carcinoma mouse model. Furthermore, we generated genetically engineered mice harboring circRNA expression constructs. We demonstrate that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. Overall, these tools expand the genetic toolkit available for the functional characterization of circRNAs of interest.

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Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris

Pharmaceuticals ◽

10.3390/ph14070698 ◽

2021 ◽

Vol 14 (7) ◽

pp. 698

Author(s):

Tina V. A. Hansen ◽

Richard K. Grencis ◽

Mohamed Issouf ◽

Cédric Neveu ◽

Claude L. Charvet

Keyword(s):

Single Dose ◽

Mouse Model ◽

Xenopus Laevis ◽

Acetylcholine Receptor ◽

Drug Target ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Trichuris Muris ◽

Electrode Voltage

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

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Molecular Characterization of the Interaction of Borrelia parkeri and Borrelia turicatae with Human Complement Regulators

Infection and Immunity ◽

10.1128/iai.00089-10 ◽

2010 ◽

Vol 78 (5) ◽

pp. 2199-2208 ◽

Cited By ~ 16

Author(s):

Melanie Schott ◽

Sonja Grosskinsky ◽

Christiane Brenner ◽

Peter Kraiczy ◽

Reinhard Wallich

Keyword(s):

Binding Capacity ◽

Functional Characterization ◽

Ectopic Expression ◽

Factor H ◽

Human Complement ◽

Related Protein ◽

Relapsing Fever ◽

Extracellular Matrix Components ◽

Complement Regulators

ABSTRACT In North America, tick-borne relapsing fever is caused by the species Borrelia hermsii, B. parkeri, and B. turicatae, which are transmitted to humans through the bite of the respective infected tick vectors. Here we describe the identification and functional characterization of a surface lipoprotein of B. parkeri, designated BpcA, that binds the human complement regulators factor H and factor H-related protein 1 and, simultaneously, the host protease plasminogen. In contrast, the homologous B. turicatae protein failed to bind human factor H and factor H-related protein 1 but retained its plasminogen binding capacity. Factor H bound to BpcA maintains its regulatory capacity to control C3b deposition and C3 convertase activity. Ectopic expression of BpcA in a serum-sensitive B. burgdorferi strain protects transformed cells from complement-mediated killing. Furthermore, bound plasminogen/plasmin endows B. parkeri and B. turicatae with the potential to degrade extracellular matrix components. These findings expand our understanding of the putative recent evolutionary separation of Borrelia parkeri and Borrelia turicatae, provide evidence that B. parkeri differs from B. turicatae in its ability to resist complement attack, and may help in understanding the pathological processes underlying tick-borne relapsing fever.

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TMEA: A Thermodynamically Motivated Framework for Functional Characterization of Biological Responses to System Acclimation

Entropy ◽

10.3390/e22091030 ◽

2020 ◽

Vol 22 (9) ◽

pp. 1030

Author(s):

Kevin Schneider ◽

Benedikt Venn ◽

Timo Mühlhaus

Keyword(s):

Free Energy ◽

Functional Characterization ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Enrichment Score ◽

Energy Investment ◽

Biological Responses ◽

Novel Approach ◽

Biological Studies

The objective of gene set enrichment analysis (GSEA) in modern biological studies is to identify functional profiles in huge sets of biomolecules generated by high-throughput measurements of genes, transcripts, metabolites, and proteins. GSEA is based on a two-stage process using classical statistical analysis to score the input data and subsequent testing for overrepresentation of the enrichment score within a given functional coherent set. However, enrichment scores computed by different methods are merely statistically motivated and often elusive to direct biological interpretation. Here, we propose a novel approach, called Thermodynamically Motivated Enrichment Analysis (TMEA), to account for the energy investment in biological relevant processes. Therefore, TMEA is based on surprisal analysis, which offers a thermodynamic-free energy-based representation of the biological steady state and of the biological change. The contribution of each biomolecule underlying the changes in free energy is used in a Monte Carlo resampling procedure resulting in a functional characterization directly coupled to the thermodynamic characterization of biological responses to system perturbations. To illustrate the utility of our method on real experimental data, we benchmark our approach on plant acclimation to high light and compare the performance of TMEA with the most frequently used method for GSEA.

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Functional characterization of a potent anti-tumor polysaccharide in a mouse model of gastric cancer

Life Sciences ◽

10.1016/j.lfs.2019.01.003 ◽

2019 ◽

Vol 219 ◽

pp. 11-19 ◽

Cited By ~ 5

Author(s):

Guangyu Li ◽

Yiran Zhang ◽

Enjun Xie ◽

Xiang Yang ◽

Hao Wang ◽

...

Keyword(s):

Gastric Cancer ◽

Mouse Model ◽

Functional Characterization

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Functional Characterization of BRASSINAZOLE-RESISTANT 1 in Panax Ginseng (PgBZR1) and Brassinosteroid Response during Storage Root Formation

International Journal of Molecular Sciences ◽

10.3390/ijms21249666 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9666

Author(s):

Hyeona Hwang ◽

Hwa-Yong Lee ◽

Hojin Ryu ◽

Hyunwoo Cho

Keyword(s):

Panax Ginseng ◽

Glycogen Synthase ◽

Functional Characterization ◽

Secondary Growth ◽

Ectopic Expression ◽

Floral Organ ◽

Membrane Receptors ◽

Storage Root ◽

Functional Conservation

Brassinosteroids (BRs) play crucial roles in the physiology and development of plants. In the model plant Arabidopsis, BR signaling is initiated at the level of membrane receptors, BRASSINOSTEROIDS INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) complex, thus activating the transcription factors (TFs) BRASSINAZOLE RESISTANT 1/BRI1-EMS-SUPPRESSOR 1 (BZR1/BES1) to coordinate BR responsive genes. BRASSINOSTEROIDS INSENSITIVE 2 (BIN2), glycogen synthase kinase 3 (GSK3) like-kinase, negatively regulates BZR1/BES1 transcriptional activity through phosphorylation-dependent cytosolic retention and shuttling. However, it is still unknown whether this mechanism is conserved in Panax ginseng C. A. Mayer, a member of the Araliaceae family, which is a shade-tolerant perennial root crop. Despite its pharmacological and agricultural importance, the role of BR signaling in the development of P. ginseng and characterization of BR signaling components are still elusive. In this study, by utilizing the Arabidopsisbri1 mutant, we found that ectopic expression of the gain of function form of PgBZR1 (Pgbzr1-1D) restores BR deficiency. In detail, ectopic expression of Pgbzr1-1D rescues dwarfism, defects of floral organ development, and hypocotyl elongation of bri1-5, implying the functional conservation of PgBZR1 in P. ginseng. Interestingly, brassinolide (BL) and BRs biosynthesis inhibitor treatment in two-year-old P. ginseng storage root interferes with and promotes, respectively, secondary growth in terms of xylem formation. Altogether, our results provide new insight into the functional conservation and potential diversification of BR signaling and response in P. ginseng.

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Ectopic expression and functional characterization of type III polyketide synthase mutants from Emblica officinalis Gaertn

Plant Cell Reports ◽

10.1007/s00299-016-2020-0 ◽

2016 ◽

Vol 35 (10) ◽

pp. 2077-2090 ◽

Cited By ~ 2

Author(s):

Girija Aiswarya ◽

Vijayanathan Mallika ◽

Luis A. J. Mur ◽

Eppurathu Vasudevan Soniya

Keyword(s):

Polyketide Synthase ◽

Functional Characterization ◽

Ectopic Expression ◽

Emblica Officinalis ◽

Type Iii Polyketide Synthase ◽

Type Iii

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Structural and Functional Characterization of Circular RNAs

10.3389/978-2-88971-928-0 ◽

2022 ◽

Keyword(s):

Functional Characterization ◽

Circular Rnas

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Anterior neurectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle

Development ◽

10.1242/dev.121.4.993 ◽

1995 ◽

Vol 121 (4) ◽

pp. 993-1004 ◽

Cited By ~ 32

Author(s):

I.L. Blitz ◽

K.W. Cho

Keyword(s):

Expression Patterns ◽

Functional Characterization ◽

Ectopic Expression ◽

Homeobox Gene ◽

Neural Induction ◽

Cement Gland ◽

Spemann's Organizer ◽

Vertical Contact

In order to study the regional specification of neural tissue we isolated Xotx2, a Xenopus homolog of the Drosophila orthodenticle gene. Xotx2 is initially expressed in Spemann's organizer and its expression is absent in the ectoderm of early gastrulae. As gastrulation proceeds, Xotx2 expression is induced in the overlying ectoderm and this domain of expression moves anteriorly in register with underlying anterior mesoderm throughout the remainder of gastrulation. The expression pattern of Xotx2 suggests that a wave of Xotx2 expression (marking anterior neurectoderm) travels through the ectoderm of the gastrula with the movement of underlying anterior (prechordal plate) mesoderm. This expression of Xotx2 is reminiscent of the Eyal-Giladi model for neural induction. According to this model, anterior neural-inducing signals emanating from underlying anterior mesoderm transiently induce anterior neural tissues after vertical contact with the overlying ectoderm. Further patterning is achieved when the ectoderm receives caudalizing signals as it comes in contact with more posterior mesoderm during subsequent gastrulation movements. Functional characterization of the Xotx2 protein has revealed its involvement in differentiation of the anterior-most tissue, the cement gland. Ectopic expression of Xotx2 in embryos induces extra cement glands in the skin as well as inducing a cement gland marker (XAG1) in isolated animal cap ectoderm. Microinjection of RNA encoding the organizer-specific homeo-domain protein goosecoid into the ventral marginal zone results in induction of the Xotx2 gene. This result, taken in combination with the indistinguishable expression patterns of Xotx2 and goosecoid in the anterior mesoderm suggests that Xotx2 is a target of goosecoid regulation.

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Functional Characterization of Pneumocystis carinii brl1 by Transspecies Complementation Analysis

Eukaryotic Cell ◽

10.1128/ec.00321-07 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2448-2452 ◽

Cited By ~ 14

Author(s):

Libera Lo Presti ◽

Moira Cockell ◽

Lorenzo Cerutti ◽

Viesturs Simanis ◽

Philippe M. Hauser

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Proteins ◽

Nuclear Membrane ◽

Opportunistic Infections ◽

Functional Characterization ◽

Pneumocystis Carinii ◽

Ectopic Expression ◽

Functional Complementation ◽

Null Alleles

ABSTRACT Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ectopic expression of this gene rescues null alleles of essential nuclear membrane proteins of the Brr6/Brl1 family in both yeasts.

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