Biology subjects

Urlaub, H.

Publications and source records attributed to Urlaub, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Structures of aberrant spliceosome intermediates on their way to disassembly

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans1. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses2,3, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-EM structures of post-Bact spliceosome intermediates from S. pombe primed for disassembly. We identify the DEAH-box helicase - G patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an over-stabilization of the U5 loop I interaction with the 5 exon leading to a single nucleotide insertion at the 5splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.

biochemistry↗

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

Site-specific metabolic labeling reveals proteome-wide phospho-dynamics

Protein phosphorylation is a critical post-translational modification that orchestrates cellular signaling. Here, we introduce PulsPhos, a method combining metabolic labeling with phosphoproteomics, spectral analysis and modeling, to quantify site-specific phosphorylation lifetimes in living cells. Phosphosite lifetimes vary over multiple orders of magnitude and are influenced by factors such as amino acid composition and subcellular localization. PulsPhos was readily applied to pharmacological perturbations revealing fundamental mechanisms governing protein phosphorylation dynamics.

molecular biology↗

bioRxiv2024

Calcium-triggered (de)ubiquitination events in synapses

Neuronal communication relies on neurotransmitter release from synaptic vesicles (SVs), whose dynamics are controlled by calcium-dependent pathways, as many thoroughly studied phosphorylation cascades. However, little is known about other post-translational modifications, as ubiquitination. To address this, we analysed resting and stimulated synaptosomes (isolated synapses) by quantitative mass spectrometry. We identified more than 5,000 ubiquitination sites on [~]2,000 proteins, the majority of which participate in SV recycling processes. Several proteins showed significant changes in ubiquitination in response to calcium influx, with the most pronounced changes in CaMKII and the clathrin adaptor protein AP180. To validate this finding, we generated a CaMKII mutant lacking the ubiquitination target site (K291) and analysed it both in neurons and non-neuronal cells. K291 ubiquitination influences CaMKII activity and synaptic function by modulating its autophosphorylation at a functionally important site (T286). We suggest that ubiquitination in response to synaptic activity is an important regulator of synaptic function.

neuroscience↗

bioRxiv2024

Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing

The eukaryotic microrchidia (MORC) protein family are DNA gyrase, Hsp90, histidine kinase, MutL (GHKL)-type ATPases involved in gene expression regulation and chromatin compaction. The molecular mechanisms underlying these activities are incompletely understood. Here we studied the full-length human MORC2 protein biochemically. We identified a DNA binding site in the C-terminus of the protein, and we observe that this region is heavily phosphorylated in cells. Phosphorylation of MORC2 reduces its affinity for DNA and appears to exclude the protein from the nucleus. We observe that DNA binding by MORC2 reduces its ATPase activity and that MORC2 can topologically entrap multiple DNA substrates between its N-terminal GHKL and C-terminal coiled coil 3 dimerization domains. Finally, we observe that the MORC2 C-terminal DNA binding region is required for gene silencing in cells. Together, our data provide a model to understand how MORC2 engages with DNA substrates to mediate gene silencing.

biochemistry↗

bioRxiv2024

mini-Complexome Profiling (mCP), an FDR-controlled workflow for global targeted detection of protein complexes

IntroductionCo-fractionation mass spectrometry couples native-like separations of protein/protein complexes with mass spectrometric proteome analysis for global characterization of protein networks. The technique allows for both de novo detection of complexes and for the detection of subtle changes in their protein composition. The typical requirement for fine-grained fractionation of >80 fractions, however, translates into significant demands on sample quantity and mass spectrometric instrument time, and represents a significant barrier to experimental replication and the use of scarce sample material (ex. Patient biopsies). MethodsWe developed mini-Complexome Profiling (mCP), a streamlined workflow with reduced requirements for fractionation and, thus, biological material and laboratory and instrument time. Soluble and membrane-associated protein complexes are extracted from biological material under mild conditions, and fractionated by Blue Native electrophoresis using commercial equipment. Each fraction is analyzed by data independent acquisition mass-spectrometry, and known protein complexes are detected based on the coelution of known components using a novel R package with a controlled false discovery rate approach. The tool is available to the community on a GitHub repository. ResultsmCP was benchmarked using HEK293 cell lysate and exhibited performance similar to established workflows, but from a significantly reduced number of fractions. We then challenged mCP by performing comparative complexome analysis of cardiomyocytes isolated from different chambers from a single mouse heart, where we identified subtle chamber-specific changes in mitochondrial OxPhos complexes. DiscussionThe reduced sample and instrument time requirements open up new applications of co-fractionation mass spectrometry, specifically for the analysis of sparse samples such as human patient biopsies. The ability to identify subtle changes between similar tissue types (left/right ventricular and atrial cardiomyocytes) serves as a proof of principle for comparative analysis of mild/asymptomatic disease states.

systems biology↗

bioRxiv2024

Plk1 inhibition delays mitotic entry revealing prophase-specific changes to the phosphoproteome

Polo-like kinase 1 (plk1) is a conserved regulator of cell division. During prophase, plk1 phosphorylates direct substrates and is involved in activation of the cyclin-dependent kinase 1 (cdk1). However, the exact functions of plk1 in prophase remain incompletely understood. By testing several cell lines and small-molecule inhibitors, we confirm that plk1 inhibition causes a delay in mitotic entry. We show that cells are delayed in a prophase-like state displaying progressively condensing chromosomes, increased microtubule dynamics, reorganization of the actin cortex, while the nuclear envelope remains intact. We show that during this prolonged prophase cdk1 activity increases gradually over several hours with individual cells stochastically reaching the entry threshold, explaining the highly variable timing of mitotic entry. We then use phosphoproteomics to characterize this prolonged prophase state revealing for the first time phosphosites specific to prophase including several regulators of chromatin organization and the cytoskeleton. Together, we show that plk1 functions as a catalyst of prophase to prometaphase transition, and by using plk1 inhibition as a tool, we identify early changes in the phosphoproteome as the cell prepares for division.

cell biology↗

bioRxiv2024

Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Delta/+ mice hearts

BackgroundPhospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14{Delta}-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14{Delta}-PLN. MethodsWe developed an improved complexome profiling (CP) workflow specifically geared towards identifying disruption of very high molecular-weight (>2 MDa) protein complexes and supercomplexes in presymptomatic R14{Delta}/+ mice hearts. Ventricular tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) proteomics analysis. Systematic analysis of CP data using conventional strategies yielded limited insights, likely due to underrepresentation of cardiac-specific complexes in the curated protein complex databases used as ground-truth for analysis. We thus developed PERCOM: a novel data analysis strategy that does not rely upon protein complex databases and can, furthermore, be implemented on widely available spreadsheet software. ResultsSEC-DIA-MS coupled with PERCOM identified 296 proteins with disrupted elution profiles in presymptomatic 28wk-old R14{Delta}/+ mice. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Alterations to mitochondrial and ICD supercomplexes were observed in mice as young as 9wks of age and were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. ConclusionUsing a novel CP workflow, we identify mitochondrial alterations as an early-stage R14{Delta}-PLN event and provide preliminary data showing effects at the ICD. These molecular components underlie critical cardiac functions and their alteration at a young age may contribute to R14{Delta}-PLN pathogenesis.

cell biology↗

bioRxiv2024

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

biochemistry↗

bioRxiv2023

Control of a type III-Dv CRISPR-Cas system by the transcription factor RpaB and interaction of its transcribed leader with the DEAD-box RNA helicase CrhR

CRISPR-Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR-Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacterium Synechocystis sp. PCC 6803 encodes three different CRISPR-Cas systems. The expression of one of these, a type III-Dv system, responds to changes in environmental conditions, such as nitrogen starvation or varying light intensities. Here, we found that the promoter of the six-gene cas operon for the type III-Dv system is controlled by the light-and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif located 53 to 70 nt upstream of the transcription start site, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, we found that the 125 nt leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled to mass spectrometry analysis revealed six residues involved in the CrhR-RNA interaction. Of these, L103, F104, H225, and C371 were predicted to be on the surface of a dimeric CrhR model, while C184 was not on the surface, and P443 could not be assigned to a structural element. These results showed that the expression of the CRISPR-Cas system is linked to the redox status of the photosynthetic cyanobacterial cell at two different levels. While RpaB affects transcription, CrhR interacts with the leader transcript posttranscription. These results highlight the complex interplay between a CRISPR-Cas system and its host cell.

microbiology↗

bioRxiv2023

Phosphoproteomics identifies targets of Mos-MAPK regulating translation and spindle organization in oocyte meiosis

The Mos kinase activates the ERK/MAPK pathway during oocyte meiosis, controlling essential meiotic functions in species across metazoa. However, despite its significance, the molecular targets of Mos-MAPK remain largely unidentified. Here, we addressed this question using starfish oocytes ideally suited to combine cellular assays with phosphoproteomics. This revealed CPE-mediated mRNA polyadenylation as a prominent target of Mos-MAPK, and we show that translation is required to drive the second meiotic division. Secondly, we identify a well-defined subset of cytoskeletal regulators as targets of Mos-MAPK. We show that this regulation is critical to ensure the asymmetry of meiotic divisions primarily by reducing the growth of astral microtubules. This allows positioning of the spindle directly beneath the cortex and prevents the separation of spindle poles in anaphase, thereby minimizing polar body size. Thus, by phosphoproteomics we reveal molecular modules controlled by Mos-MAPK explaining how this single, conserved kinase can act as a switch between the mitotic and meiotic division programs.

cell biology↗

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

Phosphorylation-dependent interactions of VAPB and ELYS contribute to the temporal progression of mitosis

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear envelope in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate the formation of novel nuclear pore complexes. Here we describe the interaction of ELYS with the membrane protein VAPB. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which is shown to mediate interaction with the MSP (major sperm protein)-domain of VAPB. Phosphorylation-dependent binding of VAPB to ELYS is demonstrated by peptide binding assays and co-immunoprecipitation experiments. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB resulted in prolonged mitosis and slow progression from meta-to anaphase and also to chromosome segregation defects. Together, our results suggest an active role of VAPB in recruiting membrane fragments to chromatin and in the biogenesis of a novel nuclear envelope during mitosis.

cell biology↗

bioRxiv2023

Phosphatases modified by LH signaling in rat ovarian follicles and their role in regulating the NPR2 guanylyl cyclase

In response to luteinizing hormone, multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-minute exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the PPP family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/544636v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2622cdorg.highwire.dtl.DTLVardef@1b6eef7org.highwire.dtl.DTLVardef@102da9borg.highwire.dtl.DTLVardef@5e3221_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary sentenceQuantitative mass spectrometric analysis of phosphatases whose phosphorylation state is rapidly modified by luteinizing hormone provides clues about how LH signaling dephosphorylates NPR2 as well as a resource for future studies.

cell biology↗

bioRxiv2023

Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics

Extremophiles, such as the hyperthermophilic archaeon Pyrococcus furiosus, thrive under extreme conditions and must rapidly adapt to changes in the physical parameters of their natural environment for short-term and long-term survival. When inhabiting hydrothermal vents, these organisms face substantial temperature gradients, necessitating the evolution of adaptive thermal stress mechanisms. However, the dynamics and coordination of cellular responses at the transcriptome and proteome levels remain underexplored. This study presents an integrated analysis of RNA-sequencing and mass spectrometry data to elucidate the transcriptomic and proteomic responses to heat and cold shock stress and recovery in P. furiosus. Our results reveal surprisingly rapid and dynamic changes in gene and protein expression patterns associated with these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, which targets a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but more sustained response. Intriguingly, cold shock at 4{degrees}C elicits distinct short-term and long-term responses at both RNA and protein levels. By conducting a cluster analysis, we identified gene sets with either congruent or contrasting trends in RNA and protein changes. Notably, these clusters represent well-separated arCOG groups and appear to be tailored to their individual cellular responses. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and provide valuable insights into the molecular adaptations that facilitate life in extreme environments.

microbiology↗

bioRxiv2023

Functional genomics identifies extension of complex N-glycans as a mechanism to evade lysis by natural killer cells

Somatic mutations can lead to the transformation of healthy cells into malignant cells and allow their evasion from immune surveillance. To uncover genes that play a role in the detection and lysis of tumor cells by natural killer (NK) cells, a B lymphoblastoid cell line was subjected to a genome-wide CRISPR screen. Among the top hits that facilitated NK evasion was SPPL3, which encodes an intramembrane protease that cleaves transmembrane glycosyltransferases in the Golgi apparatus. SPPL3-deficient cells accumulated glycosyltransferases, such as acetylglucosaminyltransferase 5 (MGAT5), and displayed increased N-glycosylation. Binding of NK receptors NKG2D and CD2 to their corresponding ligands MICB and CD58, and binding of rituximab to CD20, was disrupted by SPPL3-deletion. Inhibition of N-glycan maturation restored receptor binding and sensitivity to NK cells. To elucidate the mechanism of this resistant phenotype, a secondary CRISPR screen was performed in SPPL3-deficient cells. This screen identified glycosyltransferases that catalyze the formation of highly branched N-glycans and N-acetyl-lactosamine (LacNAc) extensions as key regulators that prevent killing. A significant enrichment of poly-LacNAc-containing tetra-antennary species was confirmed by glycoproteomic analysis. These findings provide mechanistic insight into how SPPL3 deletions have been linked to cancer.

immunology↗

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

Metamorphic proteins at the basis of human autophagy initiation and lipid transfer

Autophagy is a conserved intracellular degradation pathway that uses de novo doublemembrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length sevensubunit human autophagy initiation supercomplex and found at its core ATG13-101 and transmembrane protein ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to adopt topologically distinct folds. The slow spontaneous conversion between folds creates a rate-limiting step to regulate selfassembly of the super-complex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.

biochemistry↗