A temporal difference in the stabilization of two mRNAs with a 3' iron-responsive element during iron deficiency.

The interactions of iron regulatory proteins (IRPs) with mRNAs containing an iron-responsive element (IRE) maintain cellular iron homeostasis and coordinate it with metabolism and possibly cellular behavior. The mRNA encoding transferrin receptor-1 (TFRC, TfR1), which is a major means of iron importation, has five IREs within its 3' UTR, and IRP interactions help maintain cytosolic iron through the protection of the TfR1 mRNA from degradation. An IRE within the 3' UTR of an mRNA splice variant encoding human cell division cycle 14A (CDC14A) has the potential to coordinate the cellular iron status with cellular behavior through a similar IRP-mediated mechanism. However, the stability of the CDC14A splice variant was reported earlier to be unaffected by the cellular iron status, which suggested that the IRE is not functional. We labeled newly synthesized mRNA in HEK293 cells with 5-ethynyl uridine and found that the stability of the CDC14A variant is responsive to iron deprivation, but there are two major differences from the regulation of TfR1 mRNA stability. First, the decay of the CDC14A mRNA does not utilize the Roquin-mediated reaction that acts on the TfR1 mRNA, indicating that there is flexibility in the degradative machinery antagonized by the IRE-IRP interactions. Second, the stabilization of the CDC14A mRNA is delayed relative to the TfR1 mRNA and does not occur until IRP binding activity has been induced. The result is consistent with a hierarchy of IRP interactions in which the maintenance of cellular iron through the stabilization of the TfR1 mRNA is initially prioritized.

Roquin is a major mediator of iron-regulated changes to transferrin receptor-1 mRNA stability.

Transferrin receptor-1 (TfR1) has essential iron transport and proposed signal transduction functions. Proper TfR1 regulation is a requirement for hematopoiesis, neurological development, and the homeostasis of tissues including the intestine and muscle, while dysregulation is associated with cancers and immunodeficiency. TfR1 mRNA degradation is highly regulated, but the identity of the degradation activity remains uncertain. Here, we show with gene knockouts and siRNA knockdowns that two Roquin paralogs are major mediators of iron-regulated changes to the steady-state TfR1 mRNA level within four different cell types (HAP1, HUVEC, L-M, and MEF). Roquin is demonstrated to destabilize the TfR1 mRNA, and its activity is fully dependent on three hairpin loops within the TfR1 mRNA 3'-UTR that are essential for iron-regulated instability. We further show in L-M cells that TfR1 mRNA degradation does not require ongoing translation, consistent with Roquin-mediated instability. We conclude that Roquin is a major effector of TfR1 mRNA abundance.

Neither miR-7-5p nor miR-141-3p is a major mediator of iron-responsive transferrin receptor-1 mRNA degradation.

The transferrin receptor (TfR1) is the principal means of iron importation for most mammalian cells, and regulation of mRNA stability is a major mechanism through which TfR1 expression is controlled in response to changing intracellular iron levels. An endonuclease activity degrades the TfR1 mRNA during iron-repletion, which reduces iron importation and contributes to the restoration of homeostasis. Correct identification of the TfR1 mRNA endonuclease activity is important as it has the potential to be a pharmacological target for the treatment of several pathologies in which iron homeostasis is perturbed. A recent RNA article identified both miR-7-5p and miR-141-3p as mediators of TfR1 mRNA degradation during iron-repletion. However, the proposed TfR1 microRNA binding sites are inconsistent with several earlier studies. To better understand the discrepancy, we tested the proposed sites within an assay developed to detect changes to TfR1 mRNA stability. The complete disruption of both proposed binding sites failed to impact the assay in all cell lines tested, which include cell lines derived from mouse connective tissue (L-M), a human colon adenocarcinoma (SW480), and a human ovarian carcinoma (A2780). The overexpression of a miR-7-5p mimic also failed to decrease expression of both the endogenous TfR1 mRNA and a luciferase-TfR1 reporter under conditions in which the expression of a previously identified mir-7-5p target is attenuated. As a result, it is unlikely that the microRNAs are directly mediating iron-responsive degradation of the TfR1 mRNA as recently proposed. Instead, three short hairpin loops within the TfR1 3'-UTR are shown to be more consistent as endonuclease recognition elements.

Evaluation of the iron regulatory protein-1 interactome.

The interactions of iron regulatory proteins (IRPs) with mRNAs containing an iron-responsive element (IRE) is a major means through which intracellular iron homeostasis is maintained and integrated with cellular function. Although IRE-IRP interactions have been proposed to modulate the expression of a diverse number of mRNAs, a transcriptome analysis of the interactions that form within the native mRNA structure and cellular environment has not previously been described. An RNA-CLIP study is described here that identified IRP-1 interactions occurring within a primary cell line expressing physiologically relevant amounts of mRNA and protein. The study suggests that only a small subset of the previously proposed IREs interact with IRP-1 in situ. Identifying authentic IRP interactions is not only important to a greater understanding of iron homeostasis and its integration with cell biology but also to the development of novel therapeutics that can compensate for iron imbalances.

Transferrin receptor mRNA interactions contributing to iron homeostasis.

The transferrin receptor is the primary means of iron importation for most mammalian cells and understanding its regulatory mechanisms is relevant to hematologic, oncologic, and other disorders in which iron homeostasis is perturbed. The 3' UTR of the transferrin receptor mRNA contains an instability element that is protected from degradation during iron depletion through interactions of iron regulatory proteins (IRPs) with five iron-responsive elements (IREs). The structural features required for degradation and the site of IRP binding required for in situ protection remain unclear. An RNA-CLIP strategy is described here that identifies the predominant site of IRP-1 interaction within a nontransformed primary cell line. This approach avoided complications associated with the use of elevated concentrations of protein and/or mRNA and detected interactions within the native environment of the mRNA. A compensatory mutagenesis strategy indicates that the instability element at minimum consists of three non-IRE stem-loops that can function additively, suggesting that they are not forming one highly interdependent structure. Although the IREs are not essential for instability, they enhance instability when IRP interactions are inhibited. These results are supportive of a mechanism for a graded response to the intracellular iron resulting from a progressive loss of IRP protection.

Identification of specific inhibitors for a trypanosomatid RNA editing reaction.

Several mitochondrial mRNAs of the trypanosomatid protozoa are edited through the post-transcriptional insertion and deletion of uridylates. The reaction has provided insights into basic cellular biology and is also important as a potential therapeutic target for the diseases caused by trypanosomatid pathogens. Despite this importance, the field has been hindered by the lack of specific inhibitors that could be used as probes of the reaction mechanism or developed into novel therapeutics. In this study, an electrochemiluminescent aptamer-switch was utilized in a high-throughput screen for inhibitors of a trypanosomatid RNA editing reaction. The screen identified GW5074, mitoxantrone, NF 023, protoporphyrin IX, and D-sphingosine as inhibitors of insertion editing, with IC(50) values ranging from 1 to 3 µM. GW5074 and protoporphyrin IX are demonstrated to inhibit at or before the endonuclease cleavage that initiates editing and will be valuable biochemical probes for the early events of the in vitro reaction. Since protoporphyrin IX and sphingosine are both naturally present within the trypanosomatids, their effectiveness as in vitro inhibitors is also suggestive of the potential for in vivo modulatory roles.

An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction.

An RNA editing reaction that is both essential and specific to the trypanosomatid parasites is an attractive target for new drug development. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify new drugs, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A ruthenium labeled reporter RNA is described here that overcomes these limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter RNA consists of an RNA editing substrate linked to a streptavidin-binding aptamer that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding aptamer is only activated by an editing-induced conformational change. An electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis, and binding affinity measurements were used to characterize the reporter. The highly sensitive assay could be adapted to a broad range of RNA processing reactions.

Cis-acting elements stimulating kinetoplastid guide RNA-directed editing.

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.

Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae.

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.

Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract.

The coding sequence within several mitochondrial mRNAs of the trypanosomatid protozoa is created through editing by the precise insertion and deletion of U nucleotides. The biochemical characterisation of the editing reaction in the Leishmania genus of the trypanosomatids has been hindered by the lack of a direct in vitro assay. We describe here the first direct assay for the detection of guide RNA-directed editing mediated by a mitochondrial extract prepared from two independent isolates of Leishmania tarentolae. The assay enabled the editing activity within a L. tarentolae mitochondrial extract to be significantly enriched and will facilitate the characterisation of the editing reaction. The results suggest that the difficulty in establishing an assay for the L. tarentolae reaction was not simply a result of the catalytic machinery being limiting but rather reflected the presence of constraints on both the guide RNA and mRNA sequences.

pH dependent conformational changes within the iron responsive element.

The expression of several mRNAs related to iron importation, storage and utilization within mammalian cells are regulated through interactions of iron regulatory proteins with an iron responsive element, an RNA hairpin with a bulged C. A dimethylsulfate modification interference assay was used to demonstrate that the iron responsive element undergoes significant pH dependent conformational changes. Specifically, it was demonstrated that the phylogenetically conserved A within the hairpin loop and an intra-loop C-G base pair are highly sensitive to changes in pH. The conserved C of the bulged loop does not significantly affect the pH dependent conformational changes of the hairpin loop. These studies have structural implications for an RNA-protein interaction that is critical to mammalian iron regulation.