scholarly journals Relevance of the iron-responsive element (IRE) pseudotriloop structure for IRP1/2 binding and validation of IRE-like structures using the yeast three-hybrid system

2019 ◽  
Author(s):  
Shih-Cheng Chen ◽  
René C.L. Olsthoorn
Keyword(s):  
Mutational Analysis ◽  
Binding Modes ◽  
Rna Structures ◽  
Stem Loop ◽  
Aminolevulinate Synthase ◽  
Iron Responsive Element ◽  
Bona Fide ◽  
Responsive Element ◽  
Iron Responsive Elements ◽  
Post Transcriptional Regulation

AbstractIron-responsive-elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5′ or 3′ untranslated regions (UTRs) of mRNAs, and mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5′-CAGWGH-3′ (W = A or U and H= A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Mutational analysis of FTL IRE showed that deletion of the bulged-out U6 of the pseudotriloop does not significantly affect its binding to either IRP1 or 2, but substitution with C enhances binding of both IRPs. In addition, IRP1 was found more sensitive toward changes in the pseudotriloop-stabilizing C1-G5 base pair than IRP2, while mutation of the conserved G3 was lowering the binding of both IRPs. In comparison to FTL IRE other variant IREs, IRE of 5′-aminolevulinate synthase 2 (ALAS2), SLC40A1 (also known as Ferroportin-1), and endothelial PAS domain protein 1 (EPAS1) mRNA showed slightly higher, similar, and slightly weaker affinity for IRPs, respectively, while SLC11A2 IRE exhibited very weak binding to IRP1 and medium binding to IRP2, indicating the different binding modes of IRP1 and 2. Notably, α-Synuclein IRE showed no detectable binding to either IRP1 or 2. Our results indicate that Y3H represents a bona fide system to characterize binding between IRPs and various IRE-like structures.

scholarly journals Conservation in the Iron Responsive Element Family

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1365
Author(s):  
Karl Volz
Keyword(s):  
Translational Control ◽  
Tertiary Structure ◽  
Stem Loop ◽  
Iron Regulatory Proteins ◽  
Iron Responsive Element ◽  
Responsive Element ◽  
Iron Responsive Elements ◽  
Irp1 And Irp2 ◽  
Family Membership ◽  
Secondary And Tertiary Structure

Iron responsive elements (IREs) are mRNA stem-loop targets for translational control by the two iron regulatory proteins IRP1 and IRP2. They are found in the untranslated regions (UTRs) of genes that code for proteins involved in iron metabolism. There are ten “classic” IRE types that define the conserved secondary and tertiary structure elements necessary for proper IRP binding, and there are 83 published “IRE-like” sequences, most of which depart from the established IRE model. Here are structurally-guided discussions regarding the essential features of an IRE and what is important for IRE family membership.

Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements

1989 ◽  
Vol 9 (11) ◽  
pp. 5055-5061
Author(s):  
D J Haile ◽  
M W Hentze ◽  
T A Rouault ◽  
J B Harford ◽  
R D Klausner
Keyword(s):  
Binding Protein ◽  
Reduced Form ◽  
Scatchard Analysis ◽  
Stem Loop ◽  
Lower Affinity ◽  
Translational Repressor ◽  
High Affinity ◽  
Iron Responsive Element ◽  
Responsive Element

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

scholarly journals RNA-Based Regulation of Transcription and Translation of Aureusvirus Subgenomic mRNA1

2009 ◽  
Vol 83 (19) ◽  
pp. 10096-10105 ◽  
Author(s):  
Wei Xu ◽  
K. Andrew White
Keyword(s):  
Coat Protein ◽  
Mutational Analysis ◽  
Regulation Of Transcription ◽  
Rna Structures ◽  
Rna Sequences ◽  
Stem Loop ◽  
Translational Enhancer ◽  
Stem Loop Structure ◽  
Regulatory Strategies ◽  
Rna Interaction

ABSTRACT Cucumber leaf spot virus (CLSV) is an aureusvirus (family Tombusviridae) that has a positive-sense RNA genome encoding five proteins. During infections, CLSV transcribes two subgenomic (sg) mRNAs and the larger of the two, sg mRNA1, encodes coat protein. Here, the viral RNA sequences and structures that regulate transcription and translation of CLSV sg mRNA1 were investigated. A medium-range RNA-RNA interaction in the CLSV genome, spanning 148 nucleotides, was found to be required for the efficient transcription of sg mRNA1. Further analysis indicated that the structure formed by this interaction acted as an attenuation signal required for transcription of sg mRNA1 via a premature termination mechanism. Translation of coat protein from sg mRNA1 was determined to be facilitated by a 5′-terminal stem-loop structure in the message that resembled a tRNA anticodon stem-loop. The results from mutational analysis indicated that the 5′-terminal stem-loop mediated efficient base pairing with a 3′-cap-independent translational enhancer at the 3′ end of the message, leading to efficient translation of coat protein from sg mRNA1. Comparison of the regulatory RNA structures for sg mRNA1 of CLSV to those used by the closely related tombusviruses and certain cellular RNAs revealed interesting differences and similarities that provide evolutionary and mechanistic insights into RNA-based regulatory strategies.

scholarly journals Double-Gradient Denaturing Gradient Gel Electrophoresis Assay for Identification of L-Ferritin Iron-responsive Element Mutations Responsible for Hereditary Hyperferritinemia-Cataract Syndrome: Identification of the New Mutation C14G

2001 ◽  
Vol 47 (3) ◽  
pp. 491-497 ◽  
Author(s):  
Laura Cremonesi ◽  
Antonella Fumagalli ◽  
Nadia Soriani ◽  
Maurizio Ferrari ◽  
Sonia Levi ◽  
...  
Keyword(s):  
Serum Ferritin ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Large Population ◽  
New Mutation ◽  
Stem Loop ◽  
Iron Responsive Element ◽  
Gradient Gel Electrophoresis ◽  
Responsive Element

Abstract Background: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. Methods: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5′ untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5–15% polyacrylamide and 50–95% denaturant) to separate the homo- and heteroduplexes. Results: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 μg/L. The new mutation G14C was identified. Conclusions: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

scholarly journals Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.

1989 ◽  
Vol 9 (11) ◽  
pp. 5055-5061 ◽  
Author(s):  
D J Haile ◽  
M W Hentze ◽  
T A Rouault ◽  
J B Harford ◽  
R D Klausner
Keyword(s):  
Binding Protein ◽  
Reduced Form ◽  
Scatchard Analysis ◽  
Stem Loop ◽  
Lower Affinity ◽  
Translational Repressor ◽  
High Affinity ◽  
Iron Responsive Element ◽  
Responsive Element

The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treated with iron chelators, which decrease ferritin biosynthesis, a four- to fivefold increase in the binding activity is seen and this increase is entirely caused by an increase in high-affinity binding sites. In desferrioxamine-treated cells, the high-affinity form makes up about 50% of the total IRE-BP, whereas in hemin-treated cells, the high-affinity form makes up less than 1%. The total amount of IRE-BP in the cytosol of cells is the same regardless of the prior iron treatment of the cell. Furthermore, a mutated IRE is not able to interact with the IRE-BP in a high-affinity form but only at a single lower affinity Kd of 0.7 nM. Its interaction with the IRE-BP is insensitive to the sulfhydryl status of the protein.

scholarly journals The putative iron-responsive element in the human erythroid 5-aminolevulinate synthase mRNA mediates translational control

1993 ◽  
Vol 268 (17) ◽  
pp. 12699-12705
Author(s):  
C.R. Bhasker ◽  
G. Burgiel ◽  
B. Neupert ◽  
A. Emery-Goodman ◽  
L.C. Kühn ◽  
...  
Keyword(s):  
Translational Control ◽  
Aminolevulinate Synthase ◽  
Iron Responsive Element ◽  
Responsive Element

scholarly journals Hereditary Hyperferritinemia-Cataract Syndrome Caused by a 29-Base Pair Deletion in the Iron Responsive Element of Ferritin L-Subunit Gene

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 2084-2088 ◽  
Author(s):  
Domenico Girelli ◽  
Roberto Corrocher ◽  
Luigi Bisceglia ◽  
Oliviero Olivieri ◽  
Leopoldo Zelante ◽  
...  
Keyword(s):  
Base Pair ◽  
Genetic Disorder ◽  
Dna Amplification ◽  
Negative Control ◽  
Stem Loop ◽  
Iron Regulatory Proteins ◽  
Iron Responsive Element ◽  
Ferritin Synthesis ◽  
Base Pair Deletion ◽  
Responsive Element

Abstract Iron availability regulates ferritin synthesis posttranscriptionally by the interaction between iron-regulatory proteins (IRPs) and an iron responsive element (IRE), a stem-loop sequence located on the 5′ untranslated region of ferritin mRNA. IRPs recognize IREs as a sequence/structure motif, blocking ferritin translation. Recently, we and others independently described families with a combination of hyperferritinemia (serum L-ferritin ≥ 1,000 μg/L, without iron overload) and congenital bilateral cataract, transmitted as an autosomal-dominant trait. The molecular basis were two distinct point mutations in the highly conserved CAGUG(X) hexaloop of L-ferritin IRE on chromosome 19. A new three-generation family with a similar phenotype and a unique genotype is here reported. DNA amplification by polymerase chain reaction and sequence analysis showed a 29-base pair deletion in the L-ferritin IRE, involving the whole 5′ sequence essential to the base pairing of the IRE stem. This deletion is predicted to cause the disruption of IRE stem-loop secondary structure and the nearly complete abolition of the negative control of ferritin synthesis by IRE/IRP binding. Hereditary Hyperferritinemia-Cataract Syndrome (HHCS) appears as a new genetic disorder with a unique phenotype associated with at least four different mutations in the L-ferritin IRE. Hematologists should take into account HHCS in the differential diagnosis of unexplained hyperferritinemia.

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