Biology subjects

Ficner, R.

Publications and source records attributed to Ficner, R..

10 recordsLinked to original sources

bioRxiv2026

Queuosine glycosylation protects against stress-induced tRNA fragmentation and modulates cleavage site selection

Alongside their canonical function as adaptors in translation, tRNAs are precursors of tRNA-derived fragments that can regulate diverse aspects of gene expression. Queuosine (Q), present at position 34 of eukaryotic tRNAAsn/Asp/His/Tyr, has been implicated in suppressing tRNA fragmentation. In vertebrates, Q34 of tRNAAsp and tRNATyr is further modified by mannosylation and galactosylation, respectively, catalyzed by QTMAN and QTGAL. However, the interplay between these glycosylations and other anticodon loop modifications, and their impact on tRNA fragmentation, have remained unclear. Here, we define a modification circuit in human tRNAAsp in which Q34 stimulates DNMT2-dependent m5C38 formation, while subsequent Q34 mannosylation does not impact m5C38 installation; reciprocally, m5C38 inhibits Q34 incorporation by the tRNA-guanine transglycosylase TGT. By contrast, anticodon loop modifications of tRNATyr are installed independently, although our data support a hierarchical pathway in which TRMT5-mediated m1G37 formation precedes queuosinylation and galactosylation. Alongside demonstrating that Q34 glycosylation enhances protein synthesis, our data reveal that mannosylation of Q34 protects tRNAAsp from stress-induced cleavage, thus expanding the relevance of Q glycosylation beyond translation.

Molecular Biology↗

bioRxiv2025

Alphafold 3 guided insights into the Importin b / Importin 7 heterodimer interaction and its binding to Histone H1

The nuclear import of H1 linker histones is facilitated by a heterodimer of the transport receptors Importin{beta} (Imp{beta}) and Importin7 (Imp7). While both importins can individually interact with H1, only their preassembled hetero-dimer enables its proper binding for translocation through the nuclear pore. The interaction between Imp7 and Imp{beta} is mediated by a short stretch of residues in the C-terminal region of Imp7, which plays a key role in Imp7 activation by Imp{beta}. This interaction is allosterically regulated by Imp{beta}, finely tuning the activity of the Imp{beta}/Imp7 heterodimer. A complete model of the Imp{beta}:Imp7:H1 complex was predicted by Alphafold3 (AF3) and subsequently validated using cross-linking (X-link) data, isothermal titration calorimetry (ITC), and pull-down experiments, providing robust support for the models accuracy. This model positions the globular domain of H1 within the central cavity of Imp7, in agreement with cross-linking data. Refinement of this atomic model against a previously published cryo-EM map demonstrated significantly improved correspondence compared to the earlier interpretation, which placed the H1 globular domain within Imp{beta}. This enhanced structural consistency further substantiates the accuracy of the AI-driven prediction. Detailed analysis identified the nucleoporin-like binding (NlB) region of Imp7 as a short stretch interacting with the outer surface of Imp{beta}. This interaction mode implies that regulation of FG-binding site accessibility on Imp{beta}, mediated by the FXFG nucleoporin motives within the Imp7 NlB region, likely modulates transport pathways and/or enhances the efficiency of H1 nuclear import.

molecular biology↗

bioRxiv2025

Photo-leucine incorporation in Bacillus subtilis: Identification of transporters and of a selectivity determinant in the Leu-tRNA synthetase

The elucidation of the intricate network of proteins is essential for our understanding of cellular function. In vivo crosslinking is a powerful tool for probing these interactions, with photoactivatable amino acids representing a promising next step. Here, we evaluated the application of photo-leucine in the gram-positive model organism Bacillus subtilis. This amino acid analog is incorporated into proteins, but high concentrations inhibit the growth of B. subtilis. We investigated photo-leucine homeostasis and identified the branched-chain amino acid importers BcaP and BraB as well as the bipartite exporter AzlCD as key players of its uptake and export, respectively. Additionally, we identified a previously uncharacterized exporter, AexB, a member of the "sleeping beauty" group of EamA amino acid exporters. Expression of the aexB gene is positively regulated by the transcription factor AerB, that is normally normally inactive. Selective pressure by photo-leucine triggered the acqusition of a point mutation in aerB, allowing AerB to activate aexB expression. Mutants highly resistant to photo-leucine carried a single amino acid exchange, Ala-494 to Thr, in the leucine-tRNA synthetase LeuS. Molecular docking analysis revealed that this mutation alters the leucine-binding pocket, in a manner that still allowed binding of leucine, but not photo-leucine. While high photo-leucine incorporation is beneficial for effective crosslinking, our results suggest that B. subtilis intrinsically limits its incorporation to maintain cellular tolerance. Thus, evolutionary processes provide adaptive mechanisms to adjust amino acid analogue incorporation to tolerable levels, making photo amino acids a candidate for life cell investigation of protein struture and interactions.

microbiology↗

bioRxiv2025

The Aspergillus nidulans velvet domain containing transcription factor VeA is shuttled from cytoplasm into nucleus during vegetative growth and stays there for sexual development, but has to return into cytoplasm for asexual development

Survival of multicellular organisms requires the coordinated interplay between networks regulating gene expression and controlled intracellular transport of respective regulators. Velvet domain proteins are fungal transcription factors, which form various heterodimers and play key roles in controlling early developmental decisions towards more either asexual or sexual differentiation. VeA is the central subunit of the trimeric velvet complex VelB-VeA-LaeA, which links transcriptional to epigenetic control for the coordination of fungal developmental programs to specific secondary metabolite synthesis. Nuclear localization of the VeA bridging factor is carefully controlled in fungi. VeA carries three nuclear localization signals NLS1, NLS2 and NLS3, which all contribute to nuclear import. An additional VeA nuclear export sequence (NES) provides a shuttle function, which allows the cell to relocate VeA to the cytoplasm. VeA is nuclear during vegetative growth, but has to be exported from the nucleus to allow and promote asexual development. In contrast, progression of the sexual pathway requires continuous nuclear VeA localization. Accurate nuclear import and export control of velvet proteins is further connected to specific stability control mechanism as prerequisites for fungal development and secondary metabolism. These results illustrate the various complex mutual dependencies of velvet regulatory proteins for coordinating fungal development and secondary metabolism.

molecular biology↗

bioRxiv2024

Chemical crosslinking extends and complements UV crosslinking in analysis of RNA/DNA nucleic acid-protein interaction sites by mass spectrometry

UV (ultra-violet) crosslinking with mass spectrometry (XL-MS) has been established for identifying RNA- and DNA-binding proteins along with their domains and amino acids involved. Here, we explore chemical XL-MS for RNA-protein, DNA-protein, and nucleotide-protein complexes in vitro and in vivo. We introduce a specialized nucleotide-protein-crosslink search engine, NuXL, for robust and fast identification of such crosslinks at amino acid resolution. Chemical XL-MS complements UV XL-MS by generating different crosslink species, increasing crosslinked protein yields in vivo almost four-fold, and thus it expands the structural information accessible via XL-MS. Our workflow facilitates integrative structural modelling of nucleic acid-protein complexes and adds spatial information to the described RNA-binding properties of enzymes, for which crosslinking sites are often observed close to their cofactor-binding domains. In vivo UV and chemical XL-MS data from E. coli cells analysed by NuXL establish a comprehensive nucleic acid-protein crosslink inventory with crosslink sites at amino acid level for more than 1500 proteins. Our new workflow combined with the dedicated NuXL search engine identified RNA crosslinks that cover most RNA-binding proteins, with DNA and RNA crosslinks detected in transcriptional repressors and activators.

biochemistry↗

bioRxiv2024

Molecular basis for the interactions of eIF2β with eIF5, eIF2B, and 5MP1 and their regulation by CK2

The heterotrimeric GTPase eukaryotic translation initiation factor 2 (eIF2) delivers the initiator Met-tRNAi to the ribosomal translation preinitiation complex (PIC). eIF2{beta} has three lysine-rich repeats (K-boxes), important for binding to the GTPase-activating protein eIF5, the guanine nucleotide exchange factor eIF2B, and the regulator eIF5-mimic protein (5MP). Here, we combine X-ray crystallography with NMR to understand the molecular basis and dynamics of these interactions. The crystal structure of yeast eIF5-CTD in complex with eIF2{beta} K-box 3 reveals an extended binding site on eIF2{beta}, far beyond the K-box. We show that eIF2{beta} contains three distinct binding sites, centered on each of the K-boxes, and human eIF5, eIF2B{varepsilon}, and 5MP1 can bind to all three sites, while reducing each others affinities. Our results reveal how eIF2B speeds up the dissociation of eIF5 from eIF2-GDP to promote nucleotide exchange; and how 5MP1 can destabilize eIF5 binding to eIF2 and the PIC, to promote stringent start codon selection. All these affinities are increased by CK2 phosphomimetic mutations, highlighting the role of CK2 in both remodeling and stabilizing the translation apparatus.

biochemistry↗

bioRxiv2023

The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5 ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

microbiology↗

bioRxiv2023

Ornithine is the central intermediate in the arginine degradative pathway and its regulation in Bacillus subtilis

ABSTRACTThe Gram-positive model bacterium Bacillus subtilis is able to utilize a variety of proteinogenic and non proteinogenic amino acids as sources of carbon, energy and nitrogen. The utilization of the amino acids arginine, citrulline and ornithine is catalyzed by enzymes that are encoded in the rocABC and rocDEF operons and by the rocG gene. Expression of these genes is under control of the alternative sigma factor SigL. RNA polymerase associated to this sigma factor depends on an ATP-hydrolyzing transcription activator to initiate transcription. The RocR protein acts as transcription activator for the roc genes. In this work, we have studied the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline and ornithine. This identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that it acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to ornithine and carbamate. So far, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments we demonstrate that ornithine is the molecular inducer for RocR activity. Our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector for the transcription regulator RocR. IMPORTANCEAmino acids serve as building blocks for protein biosynthesis in each living cell but can also be used as sources of carbon, energy and nitrogen. In this work we have identified ornithine as the central player in the utilization of arginine, citrulline and ornithine in the Gram-positive bacterium B. subtilis. Ornithine is the common intermediate after the first steps of arginine and citrulline degradation. We have identified the so far uncharacterized protein RocB as the enzyme responsible for the cleavage of citrulline to ornithine and carbamate. Moreover, we demonstrate that ornithine is the molecular effector that triggers ATPase activity of the transcription factor RocR. Binding of ornithine to RocR and the subsequent hydrolysis of ATP allow a functional interaction with the alternative sigma factor SigL and subsequent transcription activation of all genes of the degradative pathway.

microbiology↗

bioRxiv2020

A rendezvous of two second messengers: The c-di-AMP receptor protein DarB controls (p)ppGpp synthesis in Bacillus subtilis

Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Most likely these proteins transduce their signal by regulatory interactions with other proteins. Here, we have investigated the function of the c-di-AMP-binding protein DarB in B. subtilis, a protein consisting of two CBS (cystathionine-beta synthase) domains. We have used an unbiased search for DarB interaction partners and identified the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. Indeed, c-di-AMP inhibits the binding of DarB to Rel. The interaction results in the Rel-dependent accumulation of pppGpp. Our results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.

microbiology↗

bioRxiv2018

Mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, are of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are only rescued by expressing wild-type nup88 and not the disease-linked mutant forms of nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

cell biology↗