Biology subjects

Joseph, D. F.

Publications and source records attributed to Joseph, D. F..

4 recordsLinked to original sources

bioRxiv2026

A negatively charged unstructured loop autoinhibits mammalian Dicer and supports fidelity of miRNA biogenesis.

Dicer RNases produce small RNAs for RNA interference (RNAi) and microRNA (miRNA) pathways. Although vertebrate Dicers share a conserved overall architecture with other eukaryotic Dicers, they are uniquely adapted for the production of gene-regulating miRNAs. We report here this adaptation extends beyond the structured core of the enzyme and relies on an intrinsically disordered region (IDR) conserved across jawed vertebrates. This negatively charged IDR supports fidelity of miRNA biogenesis while inhibiting RNAi. The IDR projects into a positively charged substrate-binding groove of Dicer and stabilizes a pre-dicing state, which effectively licenses authentic miRNA precursors. Electron cryo-microscopy shows that removal of the IDR shifts the equilibrium from the pre-dicing to the dicing state, thus increasing enzymatic activity and broadening substrate accessibility. In cells, deletion, replacement or variations of the IDR affect fidelity of microRNA biogenesis, change substrate specificity, and enhance RNAi. Altogether, our work highlights functional relevance of unstructured protein parts, which escape attention in structural analyses, and introduces an autoinhibitory IDR element mimicking negative RNA charge as a component of the molecular grammar governing IDRs in RNA-binding proteins.

molecular biology↗

bioRxiv2022

Molecular basis of indispensable accuracy of mammalian miRNA biogenesis

Mammalian Dicer is the gatekeeper into the essential gene-regulating miRNA pathway. What is committing mammalian Dicer to the miRNA pathway remains unknown. We report that Dicers highly conserved DExD/H helicase domain is the key structural element supporting accurate miRNA biogenesis. While ATPase activity of the domain is non-essential, its loss is lethal in mice. It is required during canonical miRNA biogenesis for efficient recognition, high-fidelity cleavage, and strand selection. Structure of Dicer-miRNA precursor complexes showed that the DExD/H domain acquired helicase-unrelated function defining Dicer conformations, which affect substrate loading and facilitate pre-selection of miRNA precursors. Dicer lacking the DExD/H domain favors conformations enabling reduced substrate selectivity and supporting RNA interference, a different small RNA pathway. Therefore, Dicers DExD/H domain ensures indispensable high-fidelity precursor processing of mammalian miRNAs, which constitutes a structural "mold" for adaptive miRNA evolution.

molecular biology↗

bioRxiv2021

RNA beacons for detecting the human testis-determining factor

The testis-determining factor (TDF) is an essential transcriptional protein for male differentiation in mammals, expressed along spermatids to early zygotes and, to some extent, in diverse cellular lines. In this study, we developed fluorescent biosensors capable of indicating the presence of TDF. We used in vitro evolution techniques to produce RNA aptamers that bind the recombinantly expressed HMG-box, the DNA binding domain of TDF. Bioinformatic analysis along in vitro evolution setup suggested two predominant aptamer clusters with distinctive motifs. The top ranked aptamer from each cluster, M1 and M2, showed specific binding to TDF. Aptamers were fluorescently modified as molecular beacons. Pre-steady-state kinetics indicated the beacons bind rapidly, within 50 seconds, yet M1 showed better signal to noise ratios than M2. Structural predictions of the aptamer interaction indicated that M1 is composed by three stem loops and likely interact with the HMG-box of TDF through the pocket formed by the three loops. Molecular modelling of M1 beacon shows that binding to TDF entails a conformational change of the sensor resulting in the measured fluorescence changes. To our knowledge, this is the first work describing an RNA beacon for detecting the essential TDF. Potential applications and advantages over alternative methods are provided and discussed.

synthetic biology↗

bioRxiv2019

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable and steadily present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in less than 30 seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a potential biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.

bioengineering↗