International Institute of Molecular and Cell Biology

The ubiquitin-proteasome system governselective protein turnover in all eukaryotes, and its cullin-Really Interesting New Gene (RING) ligases represent the largest class of E3 ligases. Their substrate receptors (SRs) act as the 'specificity engines' of degradation, yet their contribution to human genetic disease has only recently come into focus. In this review, we provide the first systematic catalogue of 267 SRs, of which 93 are now linked to germline disorders. We synthesise emerging mechanisms, from altered degron recognition to noncanonical SR functions, and highlight how patient variants illuminate pathways for diagnosis and therapy. By connecting proteostasis, rare-disease genetics, and targeted protein degradation, SRs emerge as central nodes with broad implications for precision medicine.

PolyA tail regulates messenger RNA (mRNA) localization, stability, and translation. PolyA length affects the durability and translational activity of both endogenously and exogenously delivered mRNAs. However, long polyA stretches can undergo recombination during amplification in bacterial plasmids, impairing the production of in vitro transcribed mRNA with long polyAs. PolyA tail segmentation with heteronucleotide spacers has recently emerged as a solution. Here, we developed segmented polyA patterns that stabilize the sequence during DNA amplification and enhance mRNA translation. We designed 15 novel genetically modified polyA variants, differing in the length, placement, and frequency of spacers, and the overall length (from ~120 to 200 nucleotides). We evaluated their stability in DNA plasmids and homogeneity, translational activity, and durability in cell culture of the resulting mRNAs, comparing them to A90 tail and other known solutions, including those from existing mRNA vaccines. Selected sequences were validated in vivo. Surprisingly, we found that even frequent heteronucleotide insertions produce functional polyA tails. The most notable enhancements in protein production were observed for a segmented tail exceeding 200 nt in length [A30(CA15)11; up to six-fold compared to mRNA with A90 tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.

Purpose. Using a three-dimensional model of a part, propose an optimal part unfolding using graph modeling and topological analysis methods; develop additional criteria for optimizing flat layouts to reduce the milling labor intensity and assembly accuracy; test the developed criteria on a practical example. Research methods. In the case under study, the research was conducted using graph modeling based on topological analysis of the product model. The research material was a 4 mm thick aluminum composite sheet with a 0.4 mm aluminum layer. Two variants of flat layouts were created based on the developed graphs. The final selection of the optimal flat layout variant was determined according to the developed optimality criteria. Results. The experience of selecting technological parameters for mechanical processing of aluminum composite sheet, which provide high processing productivity and product quality, has been generalized. It has been shown that the use of graph theory based on topological analysis of the model formalizes the process of creating variants of flat layouts. New optimality criteria are proposed, namely the total length of mechanical processing and the minimum number of parallel machining trajectories of bending grooves. Optimization should be carried out according to a priority criterion, using the rest as references for choosing between comparable variants. Scientific novelty. Criteria for optimising the flat layout when machining aluminium composites have been developed, such as: total cutting length to reduce the labour intensity of milling; minimum number of parallel trajectories for machining bending grooves. Practical value. The use of the developed criteria ensures maximum productivity of mechanical processing and maximum accuracy of assembly of products made of aluminium composites. The method of using graph theory based on the analysis of model topology can be the basis for automating the process of creating flat layouts.

Alcohol use disorder (AUD) is characterized by pathological motivation to consume alcohol and cognitive inflexibility, leading to excessive alcohol seeking and use. In this study, we investigated the molecular correlates of impaired extinction of alcohol seeking during forced abstinence using a mouse model of AUD in the automated IntelliCage social system. This model distinguished AUD-prone and AUD-resistant animals based on the presence of ≥2 or <2 criteria of AUD, respectively. We used RNA sequencing to identify genes differentially expressed in the hippocampus, a brain region implicated in alcohol motivation, seeking during abstinence, and cognitive inflexibility. Our findings revealed differences in the hippocampal genes linked to the actin cytoskeleton and synaptic function, including cofilin (Cfl), and impaired synaptic transmission in the molecular layer of the hippocampal dentate gyrus (ML-DG) in ≥2 criteria mice as compared to <2 crit animals. To complement this data, we conducted local genetic manipulations in DG. Overexpression of Cfl in the polymorphic layer of the hippocampal dentate gyrus (PoDG) inhibited ML-DG synapses, increased motivation to seek alcohol and sucrose rewards, impaired extinction of seeking, and decreased reward consumption during relapse. Reducing Cfl levels had opposite effects. We also identified three SNPs in the human CFL1 gene (rs369270402, rs2376005, rs36124259) associated with increased AUD risk and found CFL1 mRNA blood levels correlated with alcohol-related hospital admissions. Overall, our study uncovers a novel mechanism linking hippocampal Cfl expression with AUD phenotypes and identifies CFL1 polymorphisms as AUD risk factor in humans.

BackgroundAccording to data from numerous research studies and reviews, the efficiency of the oocyte slow-freezing method is believed to be compromised. Here, we attempt to challenge this notion by showing our retrospective evaluation of the efficiency of the traditional vs. successfully modified method of slow-frozen oocyte recovery compared with that of vitrified oocytes. Specifically, we compared the efficiency of a modified thawing/rehydration system applied to oocytes that had already been slow-frozen with the effects of oocyte vitrification. Moreover, we verified this comparison using chemical activation of slow-frozen vs. vitrified oocytes and parthenogenetic embryo development.ResultsTwenty-two and 73 thawing cycles of slow-frozen oocytes were performed using traditional and modified rehydration methods, respectively. For comparison, 105 warming cycles of vitrified oocytes were analyzed. The survival rate of oocytes subjected to the traditional rehydration method was 65.1%. In contrast, significantly higher ratios of 89.8% and 89.7% of oocytes survived the thawing/warming procedure performed according to the modified rehydration procedure or vitrification, respectively (P ≤ 0.0001). Clinical pregnancy and implantation rates tended to be higher after a transfer of embryos developed in the modified rehydration group vs. traditional rehydration group (33.8% and 25.5% vs. 23.5% and 13.8%, respectively) and were comparable to vitrification effects (30.1% and 26.6%). Transfer of embryos developed after modified post-thawing rehydration method resulted in 23 births with 25 healthy and one preterm baby, not significantly different from 28 births reported after oocyte vitrification. Slow-frozen oocytes that were chemically activated after the superior modified rehydration method gave similar survival (91.9% vs. 99.0%), activation (76.0% vs. 64.6%) and blastocyst rates (15.2% vs. 9.4%) in comparison with vitrified oocytes, respectively.ConclusionsThe modified post-thawing rehydration method applied to slow-frozen oocytes offers benefits in terms of higher oocyte survival, fertilization, and development or activation rates, comparable to the respective measures of vitrified oocytes and, in clinical settings, high pregnancy, implantation, and birth rates. It may bring new hope to patients who have slow-frozen oocytes stored in IVF clinics.

We introduce an updated non-redundant protein-RNA docking benchmark version 3.0 (PRDBv3.0) containing 197 test cases curated from 288 unique protein-RNA complexes available in the Protein Data Bank until July 2024. Among these, 27 are unbound-unbound (UU) type where both the binding partners are available in their unbound states, 160 are unbound-bound (UB) type where only the protein is available in unbound state and remaining 10 are bound-unbound (BU) type where only the RNA is available in unbound state. The benchmark is categorized into three classes based on the conformational flexibility of the protein interface: 117 rigid-body (R) complexes with minimal structural changes, 41 semi-flexible (S) complexes showing moderate conformational changes and 29 full-flexible (F) complexes with significant conformational changes. The current benchmark represents a 62% increase in the number of test cases compared to its previous version. Binding affinity (Kd) values for a subset of 105 protein-RNA complexes from PRDBv3.0 are catalogued along with additional experimental details to develop a comprehensive protein-RNA affinity benchmark. Moreover, a total of 255 unique RNA-binding domains, present in RNA-binding proteins, are also catalogued in this updated benchmark. PRDBv3.0 will facilitate the evaluation of both rigid-body and flexible docking methods as well as the methods that aim to predict binding affinity. The updated benchmark is freely available at http://www.csb.iitkgp.ac.in/applications/PRDBv3/PRDBv3.php.

Galleria mellonella is an established invertebrate model for studying host-pathogen interactions and immune responses. Although cytokine-analogous proteins are crucial for immune regulation in both vertebrates and invertebrates, their roles in insects remain poorly understood. This study examines the involvement of an IL-19-like protein in G. mellonella immunity, particularly in response to fungal infection and immune modulation. It uses proteomic analysis, fluorescence microscopy, flow cytometry and ELISA to determine the presence of IL-19-like protein in G. mellonella hemocytes following Conidiobolus coronatus infection and exposure to the fungal metabolites harman and norharman. It also assesses the effects of recombinant IL-19 on selected functions of wax moth hemocytes. This study provides the first proteomic confirmation of an IL-19-like protein in G. mellonella, and confirms its increased expression after C. coronatus infection. Harman and norharman treatment further elevated IL-19-like protein levels in hemocytes. Functionally, IL-19 promoted hemocyte migration, network formation, and wound healing while inhibiting ROS production but did not affect apoptosis, phagocytosis or fungal susceptibility. IL-19 also upregulated Jak1- and Jak2-like proteins, indicating its involvement in JAK-STAT signaling. Our findings highlight the evolutionary conservation of cytokine-like proteins and their immunomodulatory roles in insects. They reinforce the utility of G. mellonella as a model for cytokine research and fungal pathogenesis, and provide new insights into IL-19-like proteins in immune regulation.

Due to their small size and transparency, larval zebrafish are a useful model for whole-brain imaging. Here, we present a protocol for the visualization of phosphorylated Rps6, a marker of mechanistic target of rapamycin complex 1 (mTORC1) activity, in the zebrafish brains at 5days post fertilization (dpf), using whole-mount immunofluorescence and light-sheet microscopy. We describe steps for sample preparation, storage, staining, and imaging. This protocol can also be modified for staining with antibodies against other proteins. For complete details on the use and execution of this protocol, please refer to Doszyn etal.1.

Gametogenesis is a process in which dysfunctions lead to infertility, a growing health and social problem worldwide. In both spermatogenesis and oogenesis, post-transcriptional gene expression regulation is crucial. Essentially, all mRNAs possess non-templated poly(A) tails, whose composition and dynamics (elongation, shortening, and modifications) determine the fate of mRNA. Moreover, gametogenesis, especially oogenesis, represents a unique instance of the complexity of poly(A) tails metabolism, with oocyte-specific waves of cytoplasmic polyadenylation. In this context, we provide a comprehensive transcriptomic dataset focusing on mRNA poly(A) tail composition and dynamics in murine testes and ovaries. It consists of RNA samples isolated from wild-type and transgenic mice lacking TENT5 polymerases, which can extend poly(A) tails in the cytoplasm. TENT5 deficiencies have serious consequences. For instance, the defect of TENT5D causes infertility in humans. The data described here are generated mainly using the Oxford Nanopore Direct RNA Sequencing (DRS) method, which provides ground-truth information about mRNA molecules, including poly(A) tail length and nucleotide content. For instance, we show the prevalence of uridilated tails in testicular mRNAs.