@unpublished{14591, abstract = {Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development by controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scissin machinery in plants, but the precise roles of these proteins in this process is not fully understood. Here, we characterised the roles of Plant Dynamin-Related Proteins 2 (DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to Dynamins’ recruiters, like Endophilin and Amphiphysin, in the CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the Dsh3p1,2,3 triple-mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggests that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME. One Sentence Summary In contrast to predictions based on mammalian systems, plant Dynamin-related proteins 2 are recruited to the site of Clathrin-mediated endocytosis independently of BAR-SH3 proteins.}, author = {Gnyliukh, Nataliia and Johnson, Alexander J and Nagel, Marie-Kristin and Monzer, Aline and Hlavata, Annamaria and Isono, Erika and Loose, Martin and Friml, Jiří}, booktitle = {bioRxiv}, title = {{Role of dynamin-related proteins 2 and SH3P2 in clathrin-mediated endocytosis in plants}}, doi = {10.1101/2023.10.09.561523}, year = {2023}, } @unpublished{14644, abstract = {Transcription by RNA polymerase II (Pol II) can be repressed by noncoding RNA, including the human RNA Alu. However, the mechanism by which endogenous RNAs repress transcription remains unclear. Here we present cryo-electron microscopy structures of Pol II bound to Alu RNA, which reveal that Alu RNA mimics how DNA and RNA bind to Pol II during transcription elongation. Further, we show how domains of the general transcription factor TFIIF affect complex dynamics and control repressive activity. Together, we reveal how a non-coding RNA can regulate mammalian gene expression.}, author = {Tluckova, Katarina and Testa Salmazo, Anita P and Bernecky, Carrie A}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanism of mammalian transcriptional repression by noncoding RNA}}, doi = {10.15479/AT:ISTA:14644}, year = {2023}, } @article{12051, abstract = {Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.}, author = {Daiß, Julia L and Pilsl, Michael and Straub, Kristina and Bleckmann, Andrea and Höcherl, Mona and Heiss, Florian B and Abascal-Palacios, Guillermo and Ramsay, Ewan P and Tluckova, Katarina and Mars, Jean-Clement and Fürtges, Torben and Bruckmann, Astrid and Rudack, Till and Bernecky, Carrie A and Lamour, Valérie and Panov, Konstantin and Vannini, Alessandro and Moss, Tom and Engel, Christoph}, issn = {2575-1077}, journal = {Life Science Alliance}, keywords = {Health, Toxicology and Mutagenesis, Plant Science, Biochemistry, Genetics and Molecular Biology (miscellaneous), Ecology}, number = {11}, publisher = {Life Science Alliance}, title = {{The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans}}, doi = {10.26508/lsa.202201568}, volume = {5}, year = {2022}, } @article{12143, abstract = {MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.}, author = {Zapletal, David and Taborska, Eliska and Pasulka, Josef and Malik, Radek and Kubicek, Karel and Zanova, Martina and Much, Christian and Sebesta, Marek and Buccheri, Valeria and Horvat, Filip and Jenickova, Irena and Prochazkova, Michaela and Prochazka, Jan and Pinkas, Matyas and Novacek, Jiri and Joseph, Diego F. and Sedlacek, Radislav and Bernecky, Carrie A and O’Carroll, Dónal and Stefl, Richard and Svoboda, Petr}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {21}, pages = {4064--4079.e13}, publisher = {Elsevier}, title = {{Structural and functional basis of mammalian microRNA biogenesis by Dicer}}, doi = {10.1016/j.molcel.2022.10.010}, volume = {82}, year = {2022}, } @article{10163, abstract = {The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.}, author = {Appel, Lisa-Marie and Franke, Vedran and Bruno, Melania and Grishkovskaya, Irina and Kasiliauskaite, Aiste and Kaufmann, Tanja and Schoeberl, Ursula E. and Puchinger, Martin G. and Kostrhon, Sebastian and Ebenwaldner, Carmen and Sebesta, Marek and Beltzung, Etienne and Mechtler, Karl and Lin, Gen and Vlasova, Anna and Leeb, Martin and Pavri, Rushad and Stark, Alexander and Akalin, Altuna and Stefl, Richard and Bernecky, Carrie A and Djinovic-Carugo, Kristina and Slade, Dea}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {general physics and astronomy, general biochemistry, genetics and molecular biology, general chemistry}, number = {1}, publisher = {Springer Nature}, title = {{PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC}}, doi = {10.1038/s41467-021-26360-2}, volume = {12}, year = {2021}, } @article{7487, abstract = {Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.}, author = {López De La Oliva, Amada R. and Campos-Sandoval, José A. and Gómez-García, María C. and Cardona, Carolina and Martín-Rufián, Mercedes and Sialana, Fernando J. and Castilla, Laura and Bae, Narkhyun and Lobo, Carolina and Peñalver, Ana and García-Frutos, Marina and Carro, David and Enrique, Victoria and Paz, José C. and Mirmira, Raghavendra G. and Gutiérrez, Antonia and Alonso, Francisco J. and Segura, Juan A. and Matés, José M. and Lubec, Gert and Márquez, Javier}, issn = {20452322}, journal = {Scientific reports}, number = {1}, publisher = {Springer Nature}, title = {{Nuclear translocation of glutaminase GLS2 in human cancer cells associates with proliferation arrest and differentiation}}, doi = {10.1038/s41598-020-58264-4}, volume = {10}, year = {2020}, } @article{7580, abstract = {The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.}, author = {Bezeljak, Urban and Loya, Hrushikesh and Kaczmarek, Beata M and Saunders, Timothy E. and Loose, Martin}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {12}, pages = {6504--6549}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Stochastic activation and bistability in a Rab GTPase regulatory network}}, doi = {10.1073/pnas.1921027117}, volume = {117}, year = {2020}, } @article{15061, abstract = {The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+. Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.}, author = {Pinotsis, Nikos and Zielinska, Karolina and Babuta, Mrigya and Arolas, Joan L. and Kostan, Julius and Khan, Muhammad Bashir and Schreiner, Claudia and Testa Salmazo, Anita P and Ciccarelli, Luciano and Puchinger, Martin and Gkougkoulia, Eirini A. and Ribeiro, Euripedes de Almeida and Marlovits, Thomas C. and Bhattacharya, Alok and Djinovic-Carugo, Kristina}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {36}, pages = {22101--22112}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Calcium modulates the domain flexibility and function of an α-actinin similar to the ancestral α-actinin}}, doi = {10.1073/pnas.1917269117}, volume = {117}, year = {2020}, }