Publications Snapshots

Elevated levels of the reactive metabolite methylglyoxal recapitulate progression of Type 2 Diabetes, Moraru et al., Cell Metabolism 2018

De novo mutations in DENR disrupt neuronal development and link congenital neurological disorders to defective mRNA translation re-initiation

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Several studies have shown that type 2 diabetes progresses even if patients are under optimal glycemic control. This suggests that insulin resistance and hyperglycemia are not the root causes for the disease, and raises the question of the underlying molecular changes that drive type 2 diabetes initiation and progression. Previously it was shown that type 2 diabetes patients have elevated levels of methylglyoxal (MG), a reactive metabolite that forms adducts on proteins and alters their function. In this study we find that Drosophila knockout animals that are impaired in MG detoxification have elevated endogenous MG levels and progressively developed insulin resistance, obesity and hyperglycemia, the core characteristics of type 2 diabetes. This suggests elevated MG can recapitulate some of the core aspects of diabetes in this model organism, raising the possibility that altered MG production or detoxification could be a cause of type 2 diabetes. Future work will be needed to see if this also applies to humans. [link to press release], [pubmed]

Changes in global translation elongation or initiation rates shape the proteome via the Kozak sequence, Acevedo et al., Scientific Reports 2018

kozak_sequence

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The levels of individual proteins, such as transcription factors that control cell specification, or oncogenes that control cell proliferation, are critical for determining cellular phenotypes in health and disease. Protein levels are determined by a combination of mRNA synthesis, mRNA turnover, protein synthesis and protein turnover. Although work in the past focused on transcriptional regulation, recent work has highlighted that the step of translation – the conversion of mRNA to protein – is critical in determining protein levels in a cell. Molecular mechanisms exist that regulate the rate of translation of individual mRNAs, or classes of mRNAs, thereby ‘sculpting’ the cellular proteome. This study here identifies one such molecular mechanism that cells have in their ‘toolbox’ for determining the level at which proteins get made.

During eukaryotic translation initiation, the ribosome searches for an appropriate AUG start codon. This process takes into account the RNA sequence directly upstream and downstream of the AUG – the ‘Kozak sequence’. If the Kozak sequence is strong (e.g. accAUGg), there is a high likelihood that the ribosome will initiate translation at the AUG. Surprisingly, many transcripts in both humans and in Drosophila have weak Kozak sequences. This seems counter-intuitive at first because it suggests that millions of years of evolution have not selected the sequence around that AUG for efficient translation. One explanation could be that these particular proteins need to be present at low levels in cells, hence the Kozak sequence has been selected for low level expression. This would be a ‘constitutive’ effect that occurs in all cells in the organism and under all conditions. We find in this study that the Kozak sequence also plays an additional role that can cause expression of this class of weak-Kozak containing mRNAs to be differentially regulated in response to changes in cell signaling or stress. 

This paper finds that the balance of translation initiation versus translation elongation is important in determining overall translation efficiency. When mRNAs have a strong Kozak sequence, then translation initiation is efficient, and therefore translation elongation becomes rate-limiting. Hence, drops in translation elongation rates will cause mRNAs with strong Kozak sequences to become less expressed. In contrast, mRNAs with weak Kozak sequences have low initiation rates and hence translation elongation is not limiting. This means elongation rates can drop without having much effect on translation of mRNAs with weak Kozak sequences. Therefore, this identifies a mechanism whereby a ‘global’ drop in elongation rates will differentially affect translation of mRNAs with strong versus weak Kozak sequences thereby sculpting the proteome. [pubmed]

CycD/Cdk4 and discontinuities in Dpp signaling activate TORC1 in the Drosophila wing disc, Romero-Pozuelo J et al., Developmental Cell 2017

CycD/Cdk4 and discontinuities in Dpp signaling activate TORC1 in the Drosophila wing disc

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mTORC1 is an oncogenic kinase that powerfully drives cell growth when active. How mTORC1 activity is regulated in living tissues has not been carefully studied until now because of the lack of adequate reporters that work in situ. This paper studies for the first time the activity of mTORC1 in a growing organ during animal development using the Drosophila wing as a model system. We generate a new anti-phospho-RpS6 antibody that efficiently reports mTORC1 activity by immunostaining. Surprisingly we discovered that in the developing wing tissue, mTORC1 activity is not uniform, but patchy and controlled by the cell cycle regulator CycD/Cdk4 to peak at the G1/S transition. This indicates that CycD/Cdk4 is linking the cell cycle to the main regulator of cell growth, mTORC1. [pubmed]

DENR•MCT-1 Promotes Translation Reinitiation Downstream of uORFs to Control Tissue Growth, Schleich et al., Nature 2014

DENR•MCT-1 Promotes Translation Reinitiation Downstream of uORFs to Control Tissue Growth

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DENR and MCTS1 are oncoproteins that interact physically with each other and have been linked to neurological disorders and to cancer. Although DENR has a protein domain suggesting a role in mRNA translation initiation, the molecular functions of DENR and MTCS1 were not known. To understand the function of DENR, we generated DENR knockout Drosophila. These knockout flies die due to impaired proliferation of abdominal epithelial cells. Interestingly the phenotypes of DENR knockout flies are not observed when general translation is impaired, suggesting DENR and MCTS1 might regulate translation of a specific subset of mRNAs involved in cell proliferation.

The analysis of mRNAs that require DENR for efficient translation revealed that DENR-MCTS1 specifically facilitate the expression of mRNAs with upstream open reading frames (uORFs) that have strong Kozak sequences (stuORFs). In Drosophila, these are roughly 200 mRNAs that code for regulatory proteins and kinases involved in cell proliferation and neurobiology.

uORFs are located in the 5´untranslated region of mRNAs (5 ´UTR). Many eukaryotic transcripts (>50% in human and mouse cells) have uORFs that precede the initiation codon of the main ORF. If the uORF has a strong Kozak sequence, the scanning ribosome (coming from the 5´ cap) will select the start codon of the uORF for translation initiation. In order to allow translation of the main downstream ORF, the ribosome needs to terminate translation of the uORF and then re-initiate translation. This process likely requires recycling of all translation initiation components and re-recruitment of an initiator tRNA. Our findings showed that DENR-MCT1 specifically promote this translation reinitiation process. These findings suggested a new control system regulating the translation of a specific set of mRNAs via DENR-MCT1. [pubmed]

Identification of transcripts with short stuORFs as targets for DENR•MCTS1-dependent translation in human cells, Schleich et al., Sci Rep 2007

DENR and MCTS1 are oncoproteins that interact physically with each other and have been linked to neurological disorders and to cancer. We previously discovered that DENR and MCTS1 promote translation re-initiation in Drosophila (see above). mRNAs containing short upstream Open Reading Frames (uORFs) with strong Kozak sequences specifically require DENR and MCTS1 for their translation. To understand how DENR and MCTS1 affect human cells, we studied here which human mRNAs require DENR and MCTS1 for their translation. We found that in human cells, DENR and MCTS2 regulate translation of mRNAs with very short stuORFs, coding for just 1 or 2 amino acids. This identified circa 100 genes as putative DENR and MCTS1 translational targets. One of the mRNAs that responds most strongly to DENR or MCTS1 knockdown is that of GPR37. GPR37 affects oligodendrocyte differentiation and myelination, as well as dopamine uptake by neurons. Mutations in GPR37 have been identified in autism patients. The identification of DENR and MCTS1 target transcripts will serve as a basis for future studies aimed at understanding the mechanistic involvement of DENR and MCTS1 in diseases like cancer and autism. [pubmed]

Oxygenation and adenosine deaminase support growth and proliferation of ex vivo cultured Drosophila wing imaginal discs, Strassburger et al., Development 2017

The Drosophila wing imaginal disc has been an important model system over the past decades for discovering novel biology related to development, signaling and epithelial morphogenesis. We discovered an explant setup that supports both growth and proliferation of wing discs for 9 hours ex vivo. We found this requires both oxygenation of the tissue and adenosine deaminase activity in the medium. This is a first step towards extending the duration of ex vivo culturing to enable in the future the live imaging of this organ as it grows and develops. [pubmed]

THADA regulates the organismal balance between energy storage and heat production, Moraru et al., Developmental Cell 2017

THADA regulates the organismal balance between energy storage and heat production more

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Thyroid Adenoma Associated (THADA) is an interesting gene for many reasons. As the name implies, it was found mutated in thyroid adenomas and several other types of cancer. Genome-wide association studies (GWAS) identified THADA as one of the top genes associated with type 2 diabetes. THADA has also played a role in human evolution. It is one of the most strongly selected genes that differentiates modern humans from Neanderthal. More recently, it was amongst the most strongly selected genes as humans adapted to warm or cold regions. Surprisingly, however, the function of THADA was not known.

Using Drosophila as a model system, we discovered that THADA regulates the organismal balance between energy storage (as fat) and energy consumption (to produce heat). THADA knockout flies are obese, but produce too little heat and are cold-sensitive. We found THADA does this by regulating the ER calcium pump SERCA. Interestingly, the obesity of THADA knock-out flies was rescued by re-introducing human THADA into the flies’ genomes, suggesting that THADA has the same metabolic effect in both humans and fruit flies. This work provides the first functional and mechanistic characterization of this fascinating gene, and it provides a link between climate adaptation and genetic predisposition for obesity in humans. [pubmed]

Evidence against a role for the Parkinsonism-associated protein DJ-1 in methylglyoxal detoxification, Pfaff et al., J Biol Chem 2016

In vivo, carbonyls react with thiol and amino groups of proteins, nucleic acids and amino lipids, leading to the formation of advanced glycation end products and protein cross-links. The resulting destruction of proper protein function puts the cells under stress. DJ1, a protein associated with Parkinsonism, was previously shown to act as a protein deglycase that repaired glycated proteins. In both DJ-1 knockdown cells in culture and in DJ-1β knock-out flies, we could detect no accumulation of protein adducts caused by methylglyoxal, and no effect on the viability of cells in response to methylglyoxal. [pubmed]

De novo mutations in DENR disrupt neuronal development and link congenital neurological disorders to defective mRNA translation re-initiation, Haas et al., Cell Reports 2016

Neurological disorders such as autism are complex phenotypes with complex underlying molecular causes. Hence the discovery of mutations and genes that are linked to such diseases is useful for improving our molecular understanding of these diseases. This work discovers a de novo mutation in the gene DENR in a patient with autism. This work shows that perturbations in DENR disrupt the migration and terminal branching of cortical neurons in the mouse brain. Based on our previous work on DENR and its partner MCTS1 (see below), we find that the DENR de novo mutation found in this patient disrupts the ability of DENR to promote translation re-initiation. [pubmed]

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