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 novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

2021 ◽  
Author(s):  
Tycho Marinus ◽  
Adam B Fessler ◽  
Craig A Ogle ◽  
Danny Incarnato
Keyword(s):  
Rna Structure ◽  
Structure Prediction ◽  
Critical Role ◽  
Living Cells ◽  
Pathological Process ◽  
Rna Structures ◽  
Rna Molecules ◽  
Derived Data

Abstract Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

scholarly journals A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

2020 ◽  
Author(s):  
Tycho Marinus ◽  
Adam B. Fessler ◽  
Craig A. Ogle ◽  
Danny Incarnato
Keyword(s):  
Rna Structure ◽  
Structure Prediction ◽  
Critical Role ◽  
Living Cells ◽  
Pathological Process ◽  
Rna Structures ◽  
Rna Molecules ◽  
Derived Data

ABSTRACTDue to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art SHAPE reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

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

Methods ◽  
2016 ◽  
Vol 103 ◽  
pp. 34-48 ◽  
Author(s):  
Kyle E. Watters ◽  
Angela M Yu ◽  
Eric J. Strobel ◽  
Alex H. Settle ◽  
Julius B. Lucks
Keyword(s):  
Primer Extension ◽  
Rna Structures

scholarly journals Signaling roles of phosphoinositides in the retina

2020 ◽  
pp. jlr.TR120000806 ◽  
Author(s):  
Raju V. S. Rajala
Keyword(s):  
Cell Types ◽  
Cellular Functions ◽  
Parent Molecule ◽  
Phosphoinositide Signaling ◽  
Wide Range ◽  
The Many ◽  
And Function ◽  
Different Cell Types

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (PIs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of PI kinases and PI phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule phosphatidylinositol. PI signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane binding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIs in general, in this review, we discuss recent studies and advances in PI lipid signaling in the retina. We specifically focus on PI lipids from vertebrate (e.g. bovine, rat, mice, toad, and zebrafish) and invertebrate (e.g. drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIs revealed from animal models and human diseases, and methods to study PI levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PI-modifying enzymes/phosphatases and further unravel PI regulation and function in the different cell types of the retina.

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 Sp1-Mediated circRNA circHipk2 Regulates Myogenesis by Targeting Ribosomal Protein Rpl7

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 696
Author(s):  
Junyu Yan ◽  
Yalan Yang ◽  
Xinhao Fan ◽  
Yijie Tang ◽  
Zhonglin Tang
Keyword(s):  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
C2c12 Cells ◽  
Circular Rnas ◽  
Exon 2 ◽  
Rna Molecules ◽  
Assembly Factor ◽  
And Function

Circular RNAs (circRNAs) represent a class of covalently closed single-stranded RNA molecules that are emerging as essential regulators of various biological processes. The circRNA circHipk2 originates from exon 2 of the Hipk2 gene in mice and was reported to be involved in acute promyelocytic leukemia and myocardial injury. However, the functions and mechanisms of circHipk2 in myogenesis are largely unknown. Here, to deepen our knowledge about the role of circHipk2, we studied the expression and function of circHipk2 during skeletal myogenesis. We found that circHipk2 was mostly distributed in the cytoplasm, and dynamically and differentially expressed in various myogenesis systems in vitro and in vivo. Functionally, overexpression of circHipk2 inhibited myoblast proliferation and promoted myotube formation in C2C12 cells, whereas the opposite effects were observed after circHipk2 knockdown. Mechanistically, circHipk2 could directly bind to ribosomal protein Rpl7, an essential 60S preribosomal assembly factor, to inhibit ribosome translation. In addition, we verified that transcription factor Sp1 directly bound to the promoter of circHipk2 and affected the expression of Hipk2 and circHipk2 in C2C12 myoblasts. Collectively, these findings identify circHipk2 as a candidate circRNA regulating ribosome biogenesis and myogenesis proliferation and differentiation.

scholarly journals Insights into the secondary structural ensembles of the full SARS-CoV-2 RNA genome in infected cells

2021 ◽  
Author(s):  
Silvi Rouskin ◽  
Tammy Lan ◽  
Matthew Allan ◽  
Lauren Malsick ◽  
Stuti Khandwala ◽  
...  
Keyword(s):  
Rna Folding ◽  
Dimethyl Sulfate ◽  
Ribosome Profiling ◽  
Regulatory Sequences ◽  
Rna Structures ◽  
Infected Cells ◽  
Rna Genome ◽  
Nucleotide Resolution

Abstract SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Currently, there are no antiviral drugs with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. Like many other RNA viruses, RNA structures in coronaviruses regulate gene expression and are crucial for viral replication. Although genome and transcriptome data were recently reported, there is to date little experimental data on native RNA structures in SARS-CoV-2 and most putative regulatory sequences are functionally uncharacterized. Here we report secondary structure ensembles of the entire SARS-CoV-2 genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and the algorithm ‘detection of RNA folding ensembles using expectation–maximization’ clustering (DREEM). Our results reveal previously undescribed alternative RNA conformations across the genome, including structures of the frameshift stimulating element (FSE), a major drug target, that are drastically different from prevailing in vitro population average models. Importantly, we find that this structural ensemble promotes frameshifting rates (~40%) similar to in vivo ribosome profiling studies and much higher than the canonical minimal FSE (~20%). Overall, our result highlight the value of studying RNA folding in its native, dynamic and cellular context. The genomic structures detailed here lays the groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

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.

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