scholarly journals RNA G-quadruplex structures exist and function in vivo

2019 ◽  
Author(s):  
Xiaofei Yang ◽  
Jitender Cheema ◽  
Yueying Zhang ◽  
Hongjing Deng ◽  
Susan Duncan ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Rna Structures ◽  
Specific Sequence ◽  
Form Complex ◽  
Folding Potential ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
And Function

AbstractGuanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Here, we profiled the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We found a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determined that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analysis showed that RNA G-quadruplex folding was sufficient to regulate translation and modulate plant growth. Our study reveals the existence of RNA G-quadruplex in vivo, and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

scholarly journals A 5′ UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation

2020 ◽  
Vol 48 (17) ◽  
pp. 9822-9839
Author(s):  
Connor J Maltby ◽  
James P R Schofield ◽  
Steven D Houghton ◽  
Ita O’Kelly ◽  
Mariana Vargas-Caballero ◽  
...  
Keyword(s):  
Hek293 Cells ◽  
Leak Channel ◽  
G Quadruplex ◽  
Rna Immunoprecipitation ◽  
Quadruplex Formation ◽  
Pore Domain ◽  
Normal Cellular Function ◽  
Synaptic Remodelling

Abstract RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5′ UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.

scholarly journals Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

2015 ◽  
Author(s):  
Kyle E Watters ◽  
Angela M Yu ◽  
Eric J Strobel ◽  
Alex H Settle ◽  
Julius Lucks
Keyword(s):  
Primer Extension ◽  
Computational Techniques ◽  
Rna Structures ◽  
Cellular Functions ◽  
Rna Molecules ◽  
Cellular Defense ◽  
Nucleotide Resolution ◽  
And Function

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

scholarly journals A guide to computational methods for G-quadruplex prediction

2019 ◽  
Vol 48 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Emilia Puig Lombardi ◽  
Arturo Londoño-Vallejo
Keyword(s):  
Rna Structures ◽  
Rna Sequences ◽  
Genome Wide ◽  
G Quadruplex ◽  
Associated Functions ◽  
Potential Formation ◽  
New Methodologies

Abstract Guanine-rich nucleic acids can fold into the non-B DNA or RNA structures called G-quadruplexes (G4). Recent methodological developments have allowed the characterization of specific G-quadruplex structures in vitro as well as in vivo, and at a much higher throughput, in silico, which has greatly expanded our understanding of G4-associated functions. Typically, the consensus motif G3+N1–7G3+N1–7G3+N1–7G3+ has been used to identify potential G-quadruplexes from primary sequence. Since, various algorithms have been developed to predict the potential formation of quadruplexes directly from DNA or RNA sequences and the number of studies reporting genome-wide G4 exploration across species has rapidly increased. More recently, new methodologies have also appeared, proposing other estimates which consider non-canonical sequences and/or structure propensity and stability. The present review aims at providing an updated overview of the current open-source G-quadruplex prediction algorithms and straightforward examples of their implementation.

scholarly journals Nucleus-associated phosphorylation of Ins(1,4,5)P3 to InsP6 in Dictyostelium

1995 ◽  
Vol 312 (3) ◽  
pp. 911-917 ◽  
Author(s):  
J Van der Kaay ◽  
J Wesseling ◽  
P J M Van Haastert
Keyword(s):  
Hplc Analysis ◽  
The Other ◽  
In Vitro Experiments ◽  
Specific Sequence ◽  
Metabolic Route ◽  
And Function ◽  
Mutant Cells ◽  
Potential Substrate

Although many cells contain large amounts of InsP6, its metabolism and function is still largely unknown. In Dictyostelium lysates, the formation of InsP6 by sequential phosphorylation of inositol via Ins(3,4,6)P3 has been described [Stevens and Irvine (1990) Nature (London) 346, 580-583]; the second messenger Ins(1,4,5)P3 was excluded as a potential substrate or intermediate for InsP6 formation. However, we observed that mutant cells labelled in vivo with [3H]inositol showed altered labelling of both [3H]Ins(1,4,5)P3 and [3H]InsP6. In this report we demonstrate that Ins(1,4,5)P3 is converted into InsP6 in vitro by nucleus-associated enzymes, in addition to the previously described stepwise phosphorylation of inositol to InsP6 that occurs in the cytosol. HPLC analysis indicates that Ins(1,4,5)P3 is converted into InsP6 via sequential phosphorylation at the 3-, 6- and 2-positions. Ins[32P]P6, isolated from cells briefly labelled with [32P]Pi, was analysed using Paramecium phytase, which removes the phosphates of InsP6 in a specific sequence. The 6-position contained significantly more 32P radioactivity than the 4- or 5-positions, indicating that the 6-position is phosphorylated after the other two positions. The results from these in vivo and in vitro experiments demonstrate a metabolic route involving the phosphorylation of Ins(1,4,5)P3 via Ins(1,3,4,5)P4 and Ins(1,3,4,5,6)P5 to InsP6 in a nucleus-associated fraction of Dictyostelium cells.

scholarly journals The centromere enhancer mediates centromere activation in Schizosaccharomyces pombe.

1997 ◽  
Vol 17 (6) ◽  
pp. 3305-3314 ◽  
Author(s):  
V K Ngan ◽  
L Clarke
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromosome Segregation ◽  
Dna Sequences ◽  
Direct Evidence ◽  
Central Core ◽  
Specific Sequence ◽  
Circular Dna ◽  
Centromere Function

The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat. We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function. A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy. The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S. pombe. This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region. Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function. An S. pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores. These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Extracellular vesicles as mediators and markers of acute organ injury: current concepts

2021 ◽  
Author(s):  
Birte Weber ◽  
Niklas Franz ◽  
Ingo Marzi ◽  
Dirk Henrich ◽  
Liudmila Leppik
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Organ Injury ◽  
Isolation And Characterization ◽  
Communication Processes ◽  
Incidence And Mortality ◽  
New Biomarkers ◽  
And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

scholarly journals Therapeutic Application of Brain-Specific Angiogenesis Inhibitor 1 for Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3562
Author(s):  
Mitra Nair ◽  
Chelsea Bolyard ◽  
Tae Jin Lee ◽  
Balveen Kaur ◽  
Ji Young Yoo
Keyword(s):  
Angiogenesis Inhibitor ◽  
Suppressor Gene ◽  
Chemotherapeutic Agents ◽  
Tumor Models ◽  
In Vivo Models ◽  
Herpes Simplex Viruses ◽  
Multiple Tumor ◽  
And Function

Brain-specific angiogenesis inhibitor 1 (BAI1/ADGRB1) is an adhesion G protein-coupled receptor that has been found to play key roles in phagocytosis, inflammation, synaptogenesis, the inhibition of angiogenesis, and myoblast fusion. As the name suggests, it is primarily expressed in the brain, with a high expression in the normal adult and developing brain. Additionally, its expression is reduced in brain cancers, such as glioblastoma (GBM) and peripheral cancers, suggesting that BAI1 is a tumor suppressor gene. Several investigators have demonstrated that the restoration of BAI1 expression in cancer cells results in reduced tumor growth and angiogenesis. Its expression has also been shown to be inversely correlated with tumor progression, neovascularization, and peri-tumoral brain edema. One method of restoring BAI1 expression is by using oncolytic virus (OV) therapy, a strategy which has been tested in various tumor models. Oncolytic herpes simplex viruses engineered to express the secreted fragment of BAI1, called Vasculostatin (Vstat120), have shown potent anti-tumor and anti-angiogenic effects in multiple tumor models. Combining Vstat120-expressing oHSVs with other chemotherapeutic agents has also shown to increase the overall anti-tumor efficacy in both in vitro and in vivo models. In the current review, we describe the structure and function of BAI1 and summarize its application in the context of cancer treatment.

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