Biology subjects

Niessing, D.

Publications and source records attributed to Niessing, D..

15 recordsLinked to original sources

bioRxiv2026

The Conserved N-Terminal Extension of AtKEA1 Is Largely Dispensable for Plastid Function but Contributes to Potassium Homeostasis

Members of the K efflux antiporter (KEA) family fulfill key roles in plastids and the endomembrane system. Plants and green algae possess at least one KEA mediating K/H exchange across the plastid inner envelope (IE) membrane. Recently, IE KEAs were shown to be essential for plastid gene expression (PGE), chloroplast development, and photosynthesis. Plants lacking these antiporters exhibit reduced stromal protein synthesis and accumulation of unprocessed rRNA precursors. KEA proteins comprise a conserved monovalent cation/proton antiporter 2 (CPA2) domain and a regulatory K transport and NAD-binding (KTN) domain. IE KEAs are distinguished by an additional [~]500-amino-acid N-terminal extension containing a coiled-coil (CC) domain embedded within a largely intrinsically disordered region (IDR). Intrigued by this unusual architecture, we performed phylogenetic analyses, revealing that this N-terminal fusion arose early and has been conserved throughout the green lineage. We then investigated the oligomeric state, native distribution, and function of the N-terminal domain. Using Arabidopsis thaliana, we found that IE KEAs localize to discrete clusters within the inner envelope membrane and assemble into complexes of approximately 600 kDa. Finally, complementary approaches using a functional KEA1 variant lacking the core N-terminal domains (KEA1{Delta}N) indicate that this extension plays a regulatory rather than an essential role. Our findings uncover an evolutionarily ancient regulatory module that shapes the molecular organization and function of IE KEAs, advancing our understanding of plastid ion and pH homeostasis and plastid ribosome integrity. One-sentence summaryPlastid KEA1/2 proteins feature a unique N-terminal extension that modulates potassium transport activity in a yet unknown manner but is not essential for normal plant growth under ambient conditions.

plant biology↗

bioRxiv2026

Comprehensive Characterization of the Human Neural Stem Cell Line HNSC.100 as a Versatile Model for Neurobiological Research

Studying neural-related questions is inherently challenging due to the limited number of suitable cell models. Here, we characterize a previously reported immortalized human neural stem cell line, HNSC.100, serving as a robust model for a wide range of neurobiological research questions. The cell line expresses key neural stem cell markers, including SOX2, vimentin, nestin, and allows for efficient genetic manipulation. Furthermore, HNSC.100 cells can be differentiated into neurons, astrocytes, and oligodendrocytes, thereby covering a wide spectrum of major neural cell types. We adapted corresponding differentiation protocols and established a comprehensive panel of molecular markers to validate successful differentiation, enabling precise characterization of the resulting cell population. In addition, we provide a complete dataset of RNA expression levels for all detectable genes in HSNC.100 cells. Based on this dataset, we assembled a list of expressed genes implicated in neural disorders that can be studied with this cell line. Together, we present a detailed characterization of the HNSC.100 cell line and provide new tools and reference data to facilitate its use. This resource enables researchers to evaluate the lines suitability for specific applications and to rapidly integrate HNSC.100 cells into their experimental workflows. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/700829v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1eef4e5org.highwire.dtl.DTLVardef@1100f62org.highwire.dtl.DTLVardef@1621481org.highwire.dtl.DTLVardef@305d5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Two components of the early ASH1 mRNA transport machinery undergo PUN motif-dependent liquid-liquid-phase separation

In eukaryotes, mRNA localization is a widespread mechanism of spatial gene regulation. Amongst the best-studied examples is the directional transport of ASH1 mRNA during mitosis of the budding yeast Saccharomyces cerevisiae. ASH1 mRNA is co-transcriptionally bound by the two RNA-binding proteins She2p and Loc1p and subsequently exported. In the cytoplasm, the adapter She3p binds to She2p forming an active transport complex with the myosin motor Myo4p to mediate actin-dependent transport into the daughter cell. Loc1p stays in the nucleus where it has a second function in ribosome biogenesis. In this study, we map the interaction surface of Loc1p on She2p and observe that it does not interfere with the RNA-binding interface of She2p. Analogous to a previous report on Loc1p and RNA, we could show that also Loc1p and She2p undergo liquid-liquid phase separation (LLPS). This event is caused by electrostatic interactions and can be regulated by the phosphorylation-driven alteration of She2ps oligomeric state. Furthermore, we observed that LLPS formation only requires the PUN motifs of Loc1p and that a ternary Loc1p-RNA-She2p complex also undergoes LLPS. In summary, our findings indicate that Loc1p co-transcriptionally recruits the nuclear ASH1 mRNA-She2p complex to LLPS, while the cytoplasmic Loc1p-lacking She2p-She3p-RNA transport complex does not form such LLPS.

biochemistry↗

bioRxiv2025

Structural basis of apoptosis induction by the mitochondrial voltage dependent anion channel

The voltage-dependent anion channel (VDAC) is the main gateway for metabolites across the mitochondrial outer membrane1. In addition, VDAC oligomers have been associated with apoptosis at mitochondrial stress conditions2. However, the mechanistic and structural basis of VDACs capability to induce apoptosis pathways remains poorly understood. Here, we show with biochemical and structural methods that VDAC1 oligomerization triggers the dissociation of its N-terminal -helix (VDAC1-N) from the channel interior. We used advanced lipid nanodiscs as a tool to selectively trap VDAC1 in its canonical helix-inserted and helix-exposed state to facilitate a structural characterization of both conformations by cryo-electron microscopy. The results show that slight changes in the shape and dynamics of the VDAC1 {beta}-barrel suffice to release the N-terminal helix to the channel exterior. This conformational switch addresses the long-standing question how VDAC1 can regulate partner protein binding. To confirm this hypothesis, we performed interaction studies between VDAC1 in both conformational states and the anti-apoptotic partner protein BclxL using nuclear magnetic resonance spectroscopy and could detect binding only for the helix-exposed state. These insights enabled the X-ray structure determination of the BclxL-VDAC1-N complex at high resolution and provided atomistic details on the VDAC1-N binding mode at the BH3-groove in BclxL. Further biochemical assays showed that VDAC1-N promotes pore formation of the pro-apoptotic Bcl2 protein Bak by neutralizing BclxLs inhibitory activity. These findings suggest that stress-induced oligomerization of VDAC can trigger the exposure of its N-terminal -helix leading to the neutralization of anti-apoptotic Bcl2 proteins. This mode-of-action is reminiscent of BH3-only sensitizer Bcl2 proteins3 that are efficient inducers of Bax/Bak-mediated mitochondrial outer membrane permeabilization and ultimately apoptosis.

biophysics↗

bioRxiv2025

The yeast phosphofructokinase β-subunit has ATP-dependent RNA unwinding activity and modulates cell cycle progression

Phosphofructokinase (PFK) is a rate-limiting glycolytic enzyme that also possesses an unexplored RNA binding activity. Here, we show that the - and {beta}-subunits of yeast PFK, encoded by PFK1 and PFK2, respectively, bind hundreds of mRNAs in cells, including ones coding for proteins involved in regulation of mitotic cell cycle. Pkf2p directly binds in a Mg-ATP-dependent manner to short GA-, UC-, AU- and U-rich motifs overrepresented in its mRNA targets. Strikingly, Pfk2p displays directional 5 - 3 double-stranded RNA unwinding activity not seen with Pfk1p. Furthermore, Pfk2p dynamically associates with ribosomes and promotes translation of cell cycle genes. Consequently, pfk2{Delta}, but not pfk1{Delta}, mutant cells show severely delayed G1/S phase transition which is independent of the enzymes glycolytic activity. Our results uncovered a hidden function for the Pfk2 subunit as a translational activator of mitotic cell cycle gene transcripts possibly through energy-dependent RNA unwinding activity.

molecular biology↗

bioRxiv2024

A direct interaction between the RNA-binding proteins Staufen and Tm1-I/C regulates oskar mRNP composition and transport

In the Drosophila female germline, oskar messenger RNA is transported on microtubules from the nurse cells to the posterior pole of the oocyte, where it is translated. Transport of oskar transcripts from the nurse cells into the oocyte requires dynein, while localization of the mRNAs within the oocyte to the posterior pole is dependent upon kinesin-1. Staufen, a dsRNA-binding protein, has been shown to bind the oskar mRNA transport complex in the oocyte and inactivate dynein; however, it remains unclear how kinesin is activated. Here, using surface plasmon resonance, nuclear magnetic resonance spectroscopy and RNA imaging within egg chambers, we demonstrate that Staufen directly interacts with Tm1, a non-canonical kinesin adaptor. This work provides a molecular explanation for the previously unclear role of Staufen in oskar mRNA localization.

molecular biology↗

bioRxiv2024

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient "RNA world" PUN-like motifs may have supported the correct folding of early ribozymes.

biochemistry↗

bioRxiv2024

A consensus motif in ASH1 and further transcripts unifies several RNA motifs required for interaction with the She2p/She3p transport machinery and mRNA localization in yeast

Asymmetric localization of the ASH1 transcript is a central step in the regulation of mating type switching in Saccharomyces cerevisae and a paradigm for localized mRNAs specifically recognized by the She2p/3p transport machinery. Four RNA elements in ASH1 are known to mediate bud localization, but it remained unclear what a consensus motif of all four She2p/3p recognition sites (SRS) might look like. In this study, we derive the SRS consensus motif, which is characterized by an L-shaped, double-stranded RNA conformation with two cytosine-containing sequence motifs at conserved positions and a central asymmetric bulge. It is noteworthy that the spatial arrangement of these features can be retained, despite their altered order within the nucleotide sequence. We termed these variations "configurations" and confirmed in biochemical studies that SRS function in ASH1 elements is preserved when changing configurations. We tested other known and predicted SRS elements in target transcripts of She2p/3p by biochemical and biophysical studies and confirmed the conserved SRS function even in switched configuration. Consistently, we present the SRS instance collection, which includes further predictions of SRS instances of transcript targets of the She2p/3p translocation pathway.

cell biology↗

bioRxiv2023

PURA Syndrome-causing mutations impair PUR-domain integrity and affect P-body association

Mutations in the human PURA gene cause the neuro-developmental PURA syndrome. In contrast to several other mono-genetic disorders, almost all reported mutations in this nucleic acid binding protein result in the full disease penetrance. In this study, we observed that patient mutations across PURA impair its previously reported co-localization with processing bodies. These mutations either destroyed the folding integrity, RNA binding or dimerization of PURA. We also solved the crystal structures of the N- and C-terminal PUR domains of human PURA and combined them with molecular dynamics simulations and NMR measurements. The observed unusually high dynamics and structural promiscuity of PURA indicated that this protein is particularly susceptible to mutations impairing its structural integrity. It offers an explanation why even conservative mutations across PURA result in the full penetrance of symptoms in patients with PURA syndrome.

molecular biology↗

bioRxiv2022

Human TRMT2A methylates different components of the translation machinery and contributes to translation fidelity

Methyl-5-uridine (m5U) is one of the most abundant RNA modifications found in cytosolic tRNA. tRNA methyltransferase 2 homolog A (hTRMT2A) is the dedicated mammalian enzyme of m5U formation at tRNA position 54. However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by TRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with crosslinking experiments identified a large hTRMT2A-tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that TRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of TRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.

biochemistry↗

bioRxiv2022

Structural studies of hepatitis C virus non-structural protein-5b of genotype 4a

Hepatitis C virus genotype 4a non-structural protein-5b is an RNA-dependent RNA-polymerase responsible for the efficient virus genome replication. It has been of great interest as a drug target. The availability of the crystal structure of HCV genotype 4a NS5b would facilitate the structure-based drug design. Here we report the X-ray structure of NS5b solved at 3.1 [A] resolution, setting the groundwork for the above-described therapeutic strategy. SynopsisX-ray structure of hepatitis C virus genotype 4a non-structural protein-5b, RNA-dependent RNA-polymerase responsible for the efficient virus genome replication.

molecular biology↗

bioRxiv2022

Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies

The RNA-binding protein PURA has been implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. PURA binds both DNA and RNA and has been associated with various cellular functions. Only little is known about its main cellular roles and the molecular pathways affected upon PURA depletion. Here, we show that PURA is predominantly located in the cytoplasm, where it binds to thousands of mRNAs. Many of these transcripts change abundance in response to PURA depletion. The encoded proteins suggest a role for PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels decrease the expression of the integral P-body components LSM14A and DDX6 and strongly affect P-body formation in human cells. Furthermore, PURA knockdown results in stabilization of P-body-enriched transcripts, whereas other mRNAs decrease. Hence, reduced PURA levels, as reported in patients with PURA Syndrome, influence the formation and composition of this phase-separated RNA processing machinery. Our study proposes PURA Syndrome as a new model to study the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/479353v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@5712f9org.highwire.dtl.DTLVardef@9eb133org.highwire.dtl.DTLVardef@83b7b2org.highwire.dtl.DTLVardef@1912e5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

molecular biology↗

bioRxiv2018

Combinatorial recognition of clustered RNA elementsby a multidomain RNA-binding protein, IMP3

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

molecular biology↗

bioRxiv2018

All four double-stranded RNA binding domains of Staufen2 contribute to efficient mRNA recognition and transcript localization

Throughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. They consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2) like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. Here, we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of our findings. In summary, our data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau- family proteins of different species.

biochemistry↗