Biology subjects

Muentjes, K.

Publications and source records attributed to Muentjes, K..

7 recordsLinked to original sources

bioRxiv2026

Endosomal mRNA transport coordinates local mitochondrial bioenergetics during polar fungal growth

Mitochondrial function relies on the precise spatial coordination of protein synthesis and import. Most mitochondrial proteins are nuclear-encoded and must be supplied across varying intracellular distances. In highly polarized cells such as fungal hyphae and neurons, active long-distance mRNA transport is thought to sustain distal mitochondrial function, but its mechanistic coupling to protein import and organelle physiology is unclear. Here, we demonstrate that endosomal transport of mRNAs encoding mitochondrial proteins orchestrates local bioenergetics in infectious hyphae of Ustilago maydis. Using the subunit Atp3 of electron transport chain Complex V as a model, we uncover that the endosomal mRNA transporter Rrm4 is required for efficient mitochondrial protein import, particularly at growth poles. Loss of Rrm4 leads to defects in mitochondrial import, resulting in altered physiology. We propose that endosome-coupled mRNA transport constitutes a fundamental layer of subcellular mitochondrial homeostasis, with implications extending from fungal pathogenicity to neuronal disease. Significance StatementFungal pathogens depend on efficient polar growth to execute their infection programs. Consequently, their growing cell poles face a massive local demand of energy, which is supplied by mitochondria. Currently, it is unclear how involved proteins of the mitochondrial electron transport chain (ETC) reach these distant organelles. Here, we combine fungal genetics, metabolomics, transcriptomics and minimal invasive live-cell imaging to resolve this spatial challenge in the corn pathogen Ustilago maydis. We discover that long-distance endosomal hitchhiking of mRNAs encoding mitochondrial ETC components is essential to sustain active mitochondria at the expanding pole. Ultimately, this membrane-coupled mRNA trafficking precisely orchestrates subcellular mitochondrial function, disclosing a previously unrecognized Achilles heel for the development of novel fungicides.

microbiology↗

bioRxiv2025

Dissecting the RNA binding capacity of the multi-RRM protein Rrm4 essential for endosomal mRNA transport

RNA-binding proteins (RBPs) utilize multiple RNA-binding domains (RBDs) to engage with extensive mRNA networks. Understanding the intricate interplay of modular RBDs is essential for uncovering RBP function. Yet, how individual RBDs shape transcriptome-wide interactions remains poorly understood. Here, we dissect the roles of the three RNA recognition motifs (RRMs) in the endosomal mRNA transporter Rrm4 during polar growth of Ustilago maydis. Using a comparative mutant-based iCLIP2 approach, we disclose an extensive inventory of RRM-specific binding sites. Most binding sites are prominently governed by RRM3, however, they are not critical for function. Conversely, functionally essential binding sites are recognized by a more complex RBD interplay, involving RRM1 and/or RRM2 with partial support from RRM3. By integrating transcriptome-wide RNA binding data with transcriptomics, we pinpoint their function as regulatory RNA elements affecting mRNA abundance, linking endosomal transport to stability. The modular RNA binding of Rrm4 defines distinct RNA regulons controlling mitochondrial activity, polarity factors, and cell wall remodeling - processes critical for polar growth. These findings disclose the intricate binding modes of an RBP in vivo, emphasizing how multiple RBDs differentiate functional binding sites from accessory ones to determine mRNA fate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/636894v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1af77b6org.highwire.dtl.DTLVardef@dc7178org.highwire.dtl.DTLVardef@9d7ebforg.highwire.dtl.DTLVardef@1ec5a05_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

Microbial iCLIP2: Enhanced mapping of RNA-Protein interaction by promoting protein and RNA stability

The entire RNA lifecycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV crosslinking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus Ustilago maydis as an example. Key challenges such as inherently high RNase and protease activity in fungi were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of crosslink events and improved the identification of Rrm4 binding sites. Thus, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complex I, III and V - revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study serves as a paradigm for optimizing iCLIP2 procedures in challenging organisms or tissues under high RNase/ protease conditions.

molecular biology↗

bioRxiv2024

Deciphering the RNA-binding protein network during endosomal mRNA transport

Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis. Our findings reveal a new type of MademoiseLLE domain featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides unprecedented mechanistic insights into how structural variations in the widely distributed MLLE domain facilitates mRNA attachment during endosomal transport. SignificancePolar growing cells, such as fungal hyphae and neurons, utilize endosomes to transport mRNAs along their microtubules. But how do these mRNAs precisely attach to endosomes? Our study addresses this question by investing the key mRNA transporter, Rrm4, in a fungal model microorganism. We uncovered new features of a protein-protein interaction domain that recognizes specific short linear motifs in binding partners. While this domain resembles one found in the poly(A)-binding protein, it exhibits distinct motif recognition. Deciphering the underlying binding code unveiled new interaction partners for Rrm4. The recognition system is used to form a resilient network of RNA-binding proteins (RBPs) and their interaction partners during endosomal transport. This principle is applicable to humans, highlighting its fundamental importance.

microbiology↗

bioRxiv2021

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule- dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA- binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

microbiology↗

bioRxiv2021

Establishing regulated unconventional secretion for production of heterologous proteins

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

molecular biology↗

bioRxiv2020

Polycistronic gene expression in the model microorganism Ustilago maydis

AbtractEukaryotic microorganisms transcribe monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

microbiology↗