Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Intronless β-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements.

ABSTRACT: The intronless β-globin reporter, whose mRNA is intrinsically unstable due to the lack of introns, is a useful tool to study RNA stability elements in a heterologous transcript. Insertion of a stability element leads to the accumulation of intronless β-globin mRNA that can be visualized by conventional Northern blot analyses. In this chapter, we explain how to perform the β-globin reporter assay using the ENE (expression and nuclear retention element), a triple-helix-forming RNA stability element that protects reporter mRNA from 3'- 5' decay. A list of considerations is included for the use of ENEs as a tool to stabilize other RNAs. In this chapter, we provide a brief description of how to insert an ENE sequence into the 3'-untranslated region of an intronless β-globin reporter plasmid using basic cloning technology. Then, we provide a detailed protocol for quantitative measurements of steady-state levels of β-globin mRNA. This entails the transient transfection of mammalian cells with β-globin reporter plasmids, isolation of total cellular RNA, and detection of reporter mRNA via Northern blot. This methodology can be applied for the study of any nuclear RNA stability element using the intronless β-globin reporter.

SUBMITTER: Brown JA

PROVIDER: S-EPMC5547891 | biostudies-other | 2016

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications.

Project description:Increasing evidence points to nuclear compartmentalization as a contributing mechanism for gene regulation, yet mechanisms for compartmentalization remain unclear. In this paper, we use autonomous targeting of bacterial artificial chromosome (BAC) transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs) within an HBB BAC bias a competition between pericentric versus peripheral heterochromatin targeting toward the nuclear periphery, which correlates with increased H3K9me3 across the β-globin gene cluster and locus control region. Targeting to both heterochromatin compartments is dependent on Suv39H-mediated H3K9me3 methylation. In different chromosomal contexts, PTRs confer no targeting, targeting to pericentric heterochromatin, or targeting to the periphery. A combination of fluorescent in situ hybridization, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over hundreds of kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent lamin-associated domain. Our results demonstrate that multiple cis-elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.

| S-EPMC3857487 | biostudies-literature

Splicing promotes the nuclear export of β-globin mRNA by overcoming nuclear retention elements.

Project description:Most current models of mRNA nuclear export in vertebrate cells assume that an mRNA must have specialized signals in order to be exported from the nucleus. Under such a scenario, mRNAs that lack these specialized signals would be shunted into a default pathway where they are retained in the nucleus and eventually degraded. These ideas were based on the selective use of model mRNA reporters. For example, it has been shown that splicing promotes the nuclear export of certain model mRNAs, such as human β-globin, and that in the absence of splicing, the cDNA-derived mRNA is retained in the nucleus and degraded. Here we provide evidence that β-globin mRNA contains an element that actively retains it in the nucleus and degrades it. Interestingly, this nuclear retention activity can be overcome by increasing the length of the mRNA or by splicing. Our results suggest that contrary to many current models, the default pathway for most intronless RNAs is to be exported from the nucleus, unless the RNA contains elements that actively promote its nuclear retention.

| S-EPMC4604431 | biostudies-literature

A Kaposi's sarcoma virus RNA element that increases the nuclear abundance of intronless transcripts.

Project description:The Kaposi's sarcoma-associated herpesvirus produces a 1077 nucleotide noncoding, polyadenylated, exclusively nuclear RNA called PAN that is highly expressed in lytically infected cells. We report that PAN contains a novel post-transcriptional element essential for its abundant accumulation. The element, PAN-ENE (PAN RNA expression and nuclear retention element), increases the efficiency of 3'-end formation in vivo and is sufficient to enhance RNA abundance from an otherwise inefficiently expressed intronless beta-globin construct. The PAN-ENE does not concomitantly increase the production of encoded protein. Rather, it retains the unspliced beta-globin mRNA in the nucleus. Tethering of export factors can override the nuclear retention of the PAN-ENE, supporting a mechanism whereby the PAN-ENE blocks assembly of an export-competent mRNP. The activities of the PAN-ENE are specific to intronless constructs, since inserting the PAN-ENE into a spliced beta-globin construct has no effect on mRNA abundance and does not affect localization. This is the first characterization of a cis-acting element that increases RNA abundance of intronless transcripts but inhibits assembly of an export-competent mRNP.

| S-EPMC1142595 | biostudies-literature

Specific repression of beta-globin promoter activity by nuclear ferritin.

Project description:Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult beta-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the beta-globin promoter at -153 to -148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress beta-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the beta-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant beta-globin promoter for the -150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the -150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human beta-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.

| S-EPMC55387 | biostudies-literature

Bispidine Platform as a Tool for Studying Amide Configuration Stability.

Project description:In this work, the solution conformations of seventeen 3,7-diacyl bispidines were studied by means of NMR spectroscopy including VT NMR experiments. The acyl groups included alkyl, alkenyl, aryl, hetaryl, and ferrocene moieties. The presence of syn/anti-isomers and their ratios were estimated, and some reasons explaining experimental facts were formulated. In particular, all aliphatic and heterocyclic units in the acylic R(CO) fragments led to an increased content of the syn-form in DMSO-d6 solutions. In contrast, only the anti-form was detected in DMSO-d6 and CDCl3 in the case when R = Ph, ferrocenyl, (R)-myrtenyl. In the case of a chiral compound derived from the natural terpene myrtene, a new dynamic process was found in addition to the expected inversion around the amide N-C(O) bond. Here, rotation around the CO-C=C bond in the acylic R fragment was detected, and its energy was estimated. For this compound, ΔG for amide N-C(O) inversion was found to be equal to 15.0 ± 0.2 kcal/mol, and for the rotation around the N(CO)-C2' bond, it was equal to 15.6 ± 0.3 kcal/mol. NMR analysis of the chiral bispidine-based bis-amide was conducted for the first time. Two X-ray structures are reported. For the first time, the unique syn-form was found in the crystal of an acyclic bispidine-based bis-amide. Quantum chemical calculations revealed the unexpected mechanism for amide bond inversion. It was found that the reaction does not proceed as direct N-C(O) bond inversion in the double-chair (CC) conformation but rather requires the conformational transformation into the chair-boat (CB) form first. The amide bond inversion in the latter requires less energy than in the CC form.

| S-EPMC8779339 | biostudies-literature

Conserved CTCF insulator elements flank the mouse and human beta-globin loci.

Project description:A binding site for the transcription factor CTCF is responsible for enhancer-blocking activity in a variety of vertebrate insulators, including the insulators at the 5' and 3' chromatin boundaries of the chicken beta-globin locus. To date, no functional domain boundaries have been defined at mammalian beta-globin loci, which are embedded within arrays of functional olfactory receptor genes. In an attempt to define boundary elements that could separate these gene clusters, CTCF-binding sites were searched for at the most distal DNase I-hypersensitive sites (HSs) of the mouse and human beta-globin loci. Conserved CTCF sites were found at 5'HS5 and 3'HS1 of both loci. All of these sites could bind to CTCF in vitro. The sites also functioned as insulators in enhancer-blocking assays at levels correlating with CTCF-binding affinity, although enhancer-blocking activity was weak with the mouse 5'HS5 site. These results show that with respect to enhancer-blocking elements, the architecture of the mouse and human beta-globin loci is similar to that found previously for the chicken beta-globin locus. Unlike the chicken locus, the mouse and human beta-globin loci do not have nearby transitions in chromatin structure but the data suggest that 3'HS1 and 5'HS5 may function as insulators that prevent inappropriate interactions between beta-globin regulatory elements and those of neighboring domains or subdomains, many of which possess strong enhancers.

| S-EPMC133827 | biostudies-literature

Control of human beta-globin mRNA stability and its impact on beta-thalassemia phenotype.

Project description:Messenger RNA (mRNA) stability is a critical determinant that affects gene expression. Many pathways have evolved to modulate mRNA stability in response to developmental, physiological and/or environmental stimuli. Eukaryotic mRNAs have a considerable range of half-lives, from as short as a few minutes to as long as several days. Human globin mRNAs constitute an example of highly stable mRNAs. However, a wide variety of naturally occurring mutations that result in the clinical syndrome of thalassemia can trigger accelerated mRNA decay thus controlling mRNA quality prior to translation. Distinct surveillance mechanisms have been described as being targeted for specific defective globin mRNAs. Here, we review mRNA stability mechanisms implicated in the control of β-globin gene expression and the surveillance pathways that prevent translation of aberrant β-globin mRNAs. In addition, we emphasize the importance of these pathways in modulating the severity of the β-thalassemia phenotype.

| S-EPMC3105653 | biostudies-literature

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies.

Project description:mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5'-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA(Phe), which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.

| S-EPMC4699879 | biostudies-literature

The splicing factor SRSF3 is functionally connected to the nuclear RNA exosome for intronless mRNA decay.

Project description:The RNA exosome fulfills important functions in the processing and degradation of numerous RNAs species. However, the mechanisms of recruitment to its various nuclear substrates are poorly understood. Using Epstein-Barr virus mRNAs as a model, we have discovered a novel function for the splicing factor SRSF3 in the quality control of nuclear mRNAs. We have found that viral mRNAs generated from intronless genes are particularly unstable due to their degradation by the nuclear RNA exosome. This effect is counteracted by the viral RNA-binding protein EB2 which stabilizes these mRNAs in the nucleus and stimulates both their export to the cytoplasm and their translation. In the absence of EB2, SRSF3 participates in the destabilization of these viral RNAs by interacting with both the RNA exosome and its adaptor complex NEXT. Taken together, our results provide direct evidence for a connection between the splicing machinery and mRNA decay mediated by the RNA exosome. Our results suggest that SRSF3 aids the nuclear RNA exosome and the NEXT complex in the recognition and degradation of certain mRNAs.

| S-EPMC6110769 | biostudies-literature

β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications

Project description: Not available

| S-JCBD-201305027 | bioimages