scholarly journals STAU2 binds a Complex RNA Cargo that changes temporally with production of diverse intermediate progenitor cells during mouse corticogenesis

Development ◽  
2021 ◽  
Author(s):  
Rebecca Chowdhury ◽  
Yue Wang ◽  
Melissa Campbell ◽  
Susan Goderie ◽  
Francis Doyle ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cortical Layer ◽  
Rna Molecules ◽  
Mouse Cortex ◽  
Asymmetric Divisions ◽  
Rna Immunoprecipitation ◽  
Changes Over Time ◽  
Precise Time ◽  
Intermediate Progenitor Cell

STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time-schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was ‘stable’, present at all stages, and involved in chromosome organization, macromolecule localization, translation, and DNA repair. Another subset was ‘dynamic’, changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally-regulated RNA cargo into IPCs.

scholarly journals The Emerging Role and Promise of Circular RNAs in Obesity and Related Metabolic Disorders

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1473
Author(s):  
Mohamed Zaiou
Keyword(s):  
Rna Binding ◽  
Metabolic Diseases ◽  
Rna Binding Proteins ◽  
In Vivo Studies ◽  
Future Research ◽  
Circular Rnas ◽  
Exciting Field ◽  
Rna Molecules

Circular RNAs (circRNAs) are genome transcripts that are produced from back-splicing of specific regions of pre-mRNA. These single-stranded RNA molecules are widely expressed across diverse phyla and many of them are stable and evolutionary conserved between species. Growing evidence suggests that many circRNAs function as master regulators of gene expression by influencing both transcription and translation processes. Mechanistically, circRNAs are predicted to act as endogenous microRNA (miRNA) sponges, interact with functional RNA-binding proteins (RBPs), and associate with elements of the transcriptional machinery in the nucleus. Evidence is mounting that dysregulation of circRNAs is closely related to the occurrence of a range of diseases including cancer and metabolic diseases. Indeed, there are several reports implicating circRNAs in cardiovascular diseases (CVD), diabetes, hypertension, and atherosclerosis. However, there is very little research addressing the potential role of these RNA transcripts in the occurrence and development of obesity. Emerging data from in vitro and in vivo studies suggest that circRNAs are novel players in adipogenesis, white adipose browning, obesity, obesity-induced inflammation, and insulin resistance. This study explores the current state of knowledge on circRNAs regulating molecular processes associated with adipogenesis and obesity, highlights some of the challenges encountered while studying circRNAs and suggests some perspectives for future research directions in this exciting field of study.

scholarly journals How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

2017 ◽  
Vol 63 (4) ◽  
Author(s):  
Natalia Koralewska ◽  
Weronika Hoffmann ◽  
Maria Pokornowska ◽  
Marek Cezary Milewski ◽  
Andrea Lipinska ◽  
...  
Keyword(s):  
Rna Binding ◽  
Feedback Loops ◽  
Small Interfering Rnas ◽  
Binding Studies ◽  
Hairpin Rna ◽  
Rna Molecules ◽  
Short Rnas ◽  
Microrna Processing

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by variety of proteins are being elucidated, less is known about non-protein factors; e.g. RNA molecules, that may influence the enzyme activity. Therefore, we decided to investigate the problem of whether the RNA molecules can function not only as Dicer substrates but also as its regulators.Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding with the enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

scholarly journals Interleukin 1β Regulation of the System xc− Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR

2015 ◽  
Vol 291 (4) ◽  
pp. 1643-1651 ◽  
Author(s):  
Jingxue Shi ◽  
Yan He ◽  
Sandra J. Hewett ◽  
James A. Hewett
Keyword(s):  
Half Life ◽  
Rna Binding ◽  
Critical Role ◽  
Primary Cultures ◽  
Dependent Manner ◽  
Protein Levels ◽  
Mouse Cortex ◽  
Primary Mouse ◽  
Rna Immunoprecipitation ◽  
Mrna Half Life

System xc− is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc− in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3′-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3′-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc− activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

scholarly journals Circular RNA circDLC1 Inhibits MMP1-Mediated Liver Cancer Progression via Interaction with HuR

2020 ◽  
Author(s):  
Hailing Liu ◽  
Tian Lan ◽  
Hui Li ◽  
Lin Xu ◽  
Xing Chen ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Rna Binding ◽  
Circular Rna ◽  
Hepatoma Cells ◽  
Circular Rnas ◽  
Rna Seq ◽  
Downstream Target ◽  
Rna Immunoprecipitation

Abstract Background: N6-methyladenosine (m6A) modification has been demonstrated to be closely related to cancer progression. KIAA1429, a key component of the m6A methyltransferase complex, has recently been reported to promote hepatocellular carcinoma (HCC) progression by regulating the m6A methylation. However, the involvement of circular RNAs (circRNAs) in KIAA1429-mediated HCC progression is still unknown.Methods: RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (m6A-seq) were utilized to identify KIAA1429-regulated circRNAs. The effects of circDLC1 on proliferation and metastasis of hepatoma cells were examined in vitro and in vivo. RT-qPCR was used to measure the expression of circDLC1 in HCC tissues and hepatoma cells. RNA FISH, RIP assays and biotin-labeled RNA pull-down were used to investigate the downstream effector of circDLC1. The downstream targets of circDLC1 were identified using RNA-seq.Results: Our data demonstrated that circDLC1 was downregulated in HCC tissues and closely relevant to favorable prognosis. Overexpression of circDLC1 inhibited the proliferation and motility of hepatoma cells in vitro and in vivo, while silencing of circDLC1 played the opposite role. Mechanistic investigations revealed that circDLC1 could bind to RNA-binding protein HuR, which subsequently reduced the interaction between HuR and MMP1 mRNAs, thus inhibited the expression of MMP1, finally contributed to inhibition of HCC progression.Conclusion: Our work suggests that circDLC1, a downstream target of KIAA1429, is a promising prognostic marker for HCC patients, and the circDLC1-HuR-MMP1 axis may serve as a potential therapeutic target for HCC treatment.

scholarly journals Characterization of caspase-7 interaction with RNA

2021 ◽  
Author(s):  
Alexandre Desroches ◽  
Jean-Bernard Denault
Keyword(s):  
Cell Death ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Amino Acid Sequences ◽  
Binding Assays ◽  
Rna Molecules ◽  
Caspase 7

Apoptosis is a regulated form of cell death essential to the removal of unwanted cells. At its core, a family of cysteine peptidases named caspases cleaves key proteins allowing cell death to occur. To do so, each caspase catalytic pocket recognizes preferred amino acid sequences resulting in proteolysis, but some also use exosites to select and cleave important proteins efficaciously. Such exosites have been found in a few caspases, notably caspase-7 that has a lysine patch (K38KKK) that binds RNA which acts as a bridge to RNA-binding proteins favoring proximity between the peptidase and its substrates resulting in swifter cleavage. Although caspase-7 interaction with RNA has been identified, in-depth characterization of this interaction is lacking. In this study, using in vitro cleavage assays, we determine that RNA concentration and length affect the cleavage of RNA-binding proteins. Additionally, using binding assays and RNA sequencing, we found that caspase-7 binds RNA molecules regardless of their type, sequence, or structure. Moreover, we demonstrate that the N-terminal peptide of caspase-7 reduces the affinity of the peptidase for RNA which translates into slower cleavages of RNA-binding proteins. Finally, employing engineered heterodimers, we show that a caspase-7 dimer can use both exosites simultaneously to increase its affinity to RNA because a heterodimer with only one exosite has reduced affinity for RNA and cleavage efficacy. These findings shed light on a mechanism that furthers substrate recognition by caspases and provides potential insight into its regulation during apoptosis.

scholarly journals In vitro selection of l-DNA aptamers that bind a structured d-RNA molecule

2020 ◽  
Vol 48 (4) ◽  
pp. 1669-1680 ◽  
Author(s):  
Sougata Dey ◽  
Jonathan T Sczepanski
Keyword(s):  
In Vitro Selection ◽  
Rna Binding ◽  
Rna Aptamers ◽  
Dna Aptamers ◽  
Binding Properties ◽  
Rna Molecules ◽  
Dna And Rna ◽  
Selection Of

Abstract The development of structure-specific RNA binding reagents remains a central challenge in RNA biochemistry and drug discovery. Previously, we showed in vitro selection techniques could be used to evolve l-RNA aptamers that bind tightly to structured d-RNAs. However, whether similar RNA-binding properties can be achieved using aptamers composed of l-DNA, which has several practical advantages compared to l-RNA, remains unknown. Here, we report the discovery and characterization of the first l-DNA aptamers against a structured RNA molecule, precursor microRNA-155, thereby establishing the capacity of DNA and RNA molecules of the opposite handedness to form tight and specific ‘cross-chiral’ interactions with each other. l-DNA aptamers bind pre-miR-155 with low nanomolar affinity and high selectivity despite the inability of l-DNA to interact with native d-RNA via Watson–Crick base pairing. Furthermore, l-DNA aptamers inhibit Dicer-mediated processing of pre-miRNA-155. The sequence and structure of l-DNA aptamers are distinct from previously reported l-RNA aptamers against pre-miR-155, indicating that l-DNA and l-RNA interact with the same RNA sequence through unique modes of recognition. Overall, this work demonstrates that l-DNA may be pursued as an alternative to l-RNA for the generation of RNA-binding aptamers, providing a robust and practical approach for targeting structured RNAs.

scholarly journals Identification of an In Vitro Ribonucleoprotein Complex Between a Viroid RNA and a Phloem Protein from Cucumber Plants

2001 ◽  
Vol 14 (7) ◽  
pp. 910-913 ◽  
Author(s):  
Gustavo Gómez ◽  
Vicente Pallás
Keyword(s):  
Rna Binding ◽  
High Salt ◽  
Phloem Exudate ◽  
Binding Properties ◽  
Ribonucleoprotein Complex ◽  
Rna Molecules ◽  
Stunt Viroid ◽  
Phloem Protein ◽  
Hop Stunt Viroid

We used the interaction of Hop stunt viroid (HSVd) and cucumber plants to investigate the involvement of phloem proteins in the systemic transport of RNA molecules. A ribonucleoprotein complex, stable even at high salt and temperature conditions, was detected in vitro between HSVd-RNA and the phloem exudate obtained from sectioned internodes from cucumber plants. The phloem protein 2 was recovered from this ribonucleoprotein complex and its RNA-binding properties as demonstrated by gel retardation analysis. The involvement of this protein in the movement of RNAs in cucumber is discussed.

scholarly journals Validation and classification of RNA binding proteins identified by mRNA interactome capture

2021 ◽  
Author(s):  
Vaishali ◽  
Lyudmila Dimitrova-Paternoga ◽  
Kevin Haubrich ◽  
Mai Sun ◽  
Anne Ephrussi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Modular Organization ◽  
Binding Domains ◽  
Mammalian Cell Lines ◽  
Rna Immunoprecipitation ◽  
Rna Binding Domains ◽  
Rna Binders

AbstractRNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1’s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

scholarly journals Fuzzy RNA-recognition by the Trypanosoma brucei editosome

2021 ◽  
Author(s):  
H. Ulrich Göringer ◽  
W.-Matthias Leeder
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Complex ◽  
Rna Binding ◽  
Solvent Accessibility ◽  
Binary Interaction ◽  
Rna Recognition ◽  
Rna Motifs ◽  
Rna Molecules ◽  
Ribonucleoprotein Complexes

The recognition of RNA-molecules by proteins and protein complexes is a critical step on all levels of gene expression. Typically, the generated ribonucleoprotein complexes rely on the binary interaction of defined RNA-sequences or precisely folded RNA-motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA-molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome we identified multiple similar but non-identical RNA-motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.523 (λ2). We establish that a synthetic 4V(3E)-RNA is sufficient to compete for the editosomal pre-mRNA binding site and that it is able to inhibit RNA-editing in vitro. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA-folding space by recognizing a fuzzy continuum of RNA-folds that fit a consensus graph-descriptor. This provides a mechanism on how the protein complex is able to bind the structurally pleomorphic pool of pre- and partially edited mRNAs. We speculate that other fuzzy RNA-recognition motifs exist especially for proteins that interact with multiple RNA-species.

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