Mihaela Enculescu

Bio: Mihaela Enculescu is an academic researcher from Institute of Molecular Biotechnology. The author has contributed to research in topics: Exon & RNA splicing. The author has an hindex of 2, co-authored 4 publications receiving 55 citations.

Modelling Systemic Iron Regulation during Dietary Iron Overload and Acute Inflammation: Role of Hepcidin-Independent Mechanisms.

Abstract: Systemic iron levels must be maintained in physiological concentrations to prevent diseases associated with iron deficiency or iron overload. A key role in this process plays ferroportin, the only known mammalian transmembrane iron exporter, which releases iron from duodenal enterocytes, hepatocytes, or iron-recycling macrophages into the blood stream. Ferroportin expression is tightly controlled by transcriptional and post-transcriptional mechanisms in response to hypoxia, iron deficiency, heme iron and inflammatory cues by cell-autonomous and systemic mechanisms. At the systemic level, the iron-regulatory hormone hepcidin is released from the liver in response to these cues, binds to ferroportin and triggers its degradation. The relative importance of individual ferroportin control mechanisms and their interplay at the systemic level is incompletely understood. Here, we built a mathematical model of systemic iron regulation. It incorporates the dynamics of organ iron pools as well as regulation by the hepcidin/ferroportin system. We calibrated and validated the model with time-resolved measurements of iron responses in mice challenged with dietary iron overload and/or inflammation. The model demonstrates that inflammation mainly reduces the amount of iron in the blood stream by reducing intracellular ferroportin transcription, and not by hepcidin-dependent ferroportin protein destabilization. In contrast, ferroportin regulation by hepcidin is the predominant mechanism of iron homeostasis in response to changing iron diets for a big range of dietary iron contents. The model further reveals that additional homeostasis mechanisms must be taken into account at very high dietary iron levels, including the saturation of intestinal uptake of nutritional iron and the uptake of circulating, non-transferrin-bound iron, into liver. Taken together, our model quantitatively describes systemic iron metabolism and generated experimentally testable predictions for additional ferroportin-independent homeostasis mechanisms.

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis

Abstract: Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer. Alternative splicing is a critical step in eukaryotic gene expression but its molecular rules are not fully understood. Here, the authors develop a high-throughput mutagenesis approach to comprehensively characterise determinants of alternative splicing for the RON proto-oncogene.

High-throughput mutagenesis identifies mutations and RNA binding proteins controlling CD19 splicing and CART-19 therapy resistance

Abstract: During CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy. HighlightsO_LIMutations in relapsed CART-19 patients lead to CD19 mis-splicing C_LIO_LIHigh-throughput mutagenesis uncovers ~200 single point mutations with a potential role in CART-19 therapy resistance C_LIO_LIMany mutations generate non-functional CD19 proteins by activating cryptic splice sites C_LIO_LIRNA-binding proteins such as PTBP1 are key to the expression of properly spliced, CART-19 immunotherapy-sensitive isoforms C_LI

Exon definition facilitates reliable control of alternative splicing

Abstract: Alternative splicing is a key step in eukaryotic gene expression that allows the production of multiple protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. Using mechanistic mathematical modeling, we show that alternative splicing control is facilitated if spliceosomes recognize exons as functional units (‘exon definition’). We find that exon definition is crucial to prevent the accumulation of partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-mRNA. These predictions of our model are qualitatively and quantitatively supported by high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON (MST1R). Our analysis suggests that exon definition has evolved as the dominant splice-regulatory mechanism in higher organisms to promote robust and reliable splicing outcomes. One Sentence Summary Exon definition is required for alternative precise splicing control without accumulation of undesired retention isoforms.

The Causes and Consequences of Genetic Interactions (Epistasis)

Abstract: The same mutation can have different effects in different individuals. One important reason for this is that the outcome of a mutation can depend on the genetic context in which it occurs. This dep...

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

Abstract: Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma.

Abstract: RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identified hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma. RNA G-quadruplexes (RG4s) have been functionally linked to cancer gene expression. Here, Herviou, Le Bras et al. have identified the protein machinery modulating RG4s and reveal the role and mechanism of hnRNP H/F and DHX36 in RG4-mediated translational regulation affecting cancer treatment in glioblastoma.