Biology subjects

Rappsilber, J.

Publications and source records attributed to Rappsilber, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Multi-omic analyses reveal a differential contribution of chromatin-associated PP1 holoenzymes to mitotic exit and G1 re-establishment.

Mitotic exit is an important part of the cell cycle that requires coordination of many chromatin and cytoskeleton remodelling events to successfully complete cell division and maintain cell identity. Protein de-phosphorylation is a key step in directing mitotic exit and Protein phosphatase (PP1) is essential to this process, however the specific contribution of its numerous targeting subunits is still unknown. Here we have investigated the function of three chromatin-associated PP1 targeting subunits in exiting mitosis Repo-Man, Ki-67 and PNUTS. We have generated endogenously tagged, auxin-degradable alleles for each subunit and used a multi-omic approach to address their specific contribution towards transcription resumption, chromatin accessibility and protein phosphorylation at the transition from mitosis to G1. This approach has identified their distinct role in mitotic exit, provided unique datasets for the cell cycle community, and highlighted novel functions for Ki-67 and Repo-Man in genome stability and organisation.

cell biology↗

bioRxiv2024

Molecular basis for condensin enrichment at pericentromeres

Faithful chromosome segregation requires packaging of the genome on both global and local scales. Condensin plays a crucial role at pericentromeres to resist spindle forces and ensure the bioriented attachment of kinetochores to microtubules in mitosis. Here we demonstrate that budding yeast condensin is recruited to pericentromeres through a direct interaction between its Ycg1 subunit and the pericentromeric adaptor protein, shugoshin (Sgo1). We identify a Short Linear Motif (SLiM), termed CR1, within the C-terminal region of Sgo1 which inserts into a conserved pocket on Ycg1. Disruption of this interface abolishes the Sgo1-condensin interaction, prevents condensin recruitment to pericentromeres and results in defective sister kinetochore biorientation in mitosis. Similar motifs to CR1 are found in known and potential condensin binding partners and the Ycg1 binding pocket is broadly conserved, including in the mammalian homolog CAP-G. Overall, we uncover the molecular mechanism that targets condensin to define a specialized chromosomal domain.

cell biology↗

bioRxiv2024

Molecular basis of mRNA delivery to the bacterial ribosome

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

biochemistry↗

bioRxiv2024

PLK1-Mediated Phosphorylation Cascade Activates the Mis18 Complex to Ensure Centromere Inheritance

Accurate chromosome segregation requires the attachment of spindle microtubules to centromeres, which are epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, existing CENP-A nucleosomes undergo dilution as they get redistributed among the two DNA strands. To preserve centromere identity, CENP-A levels must be restored in a cell-cycle controlled manner orchestrated by the Mis18 complex. Here we provide a comprehensive mechanistic basis for PLK1-mediated licensing of CENP-A loading. We demonstrate that PLK1 interacts with Mis18 and Mis18BP1 subunits of the Mis18 complex by recognising self-primed phosphorylations of Mis18 (S54) and Mis18BP1 (T78 and S93) through its Polo-box binding domain. Disrupting these PLK1 phosphorylations perturbed the centromere recruitment of HJURP and new CENP-A loading. Biochemical and functional analyses show that phosphorylation of Mis18 and subsequent PLK1 binding is required to activate the Mis18/{beta} complex for robust Mis18/{beta}-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance. One-Sentence SummaryPLK1 phosphorylation cascade licenses CENP-A loading by facilitating HJURP centromere recruitment via Mis18/{beta} activation.

cell biology↗

bioRxiv2023

RNA 5' terminal nucleotide determines the strength of the RIG-I/IFN signaling pathway

The interferon (IFN) response is crucial for antiviral activity, but its overstimulation can lead to a wide range of autoimmune disorders. The cytoplasmic pattern recognition receptor RIG-I detects viral double-stranded RNAs (dsRNAs) and endogenous polymerase III transcripts carrying a 5'-triphosphate (5'-ppp) or 5'-diphosphate (5'-pp) moiety, triggering phosphorylation of IRF3 and IFN immune response. While many viral RNAs initiate with 5'-ppp-adenosine (5'-pppA) and most endogenous Pol III transcripts in higher eukaryotes start with 5'-ppp-guanosine (5'-pppG), no apparent reason for this bias has been identified so far. Here we demonstrate that dsRNAs initiating with 5'-pppA trigger stronger RIG-I/IFN response than those starting with 5'-pppG. We show that several GTP-binding proteins interact preferentially with 5'-pppG RNAs. Finally, supplementation with guanosine, but not adenosine, which rapidly increases intracellular concentrations of GTP and ATP, respectively, eliminates the difference in immunogenicity between 5'-pppG and 5'-pppA RNAs. Our findings suggest that 5'-pppG RNAs may enable certain RNA viruses and Pol III transcripts to limit detection by innate immune receptors. These results offer new insights into the sequence-dependent activation of the RIG-I/IFN pathway and have important implications for both antiviral immunity and the role of Pol III-derived RNAs in autoimmune diseases.

molecular biology↗

bioRxiv2023

Rescuing error control in crosslinking mass spectrometry

Crosslinking mass spectrometry is a powerful tool for studying protein-protein interactions under native or near native conditions in complex mixtures. By help of novel search controls, we show that measures that aim to improve the number of identifications based on heuristic considerations can undermine error estimation in non-obvious ways. The relationship between decoys and false positives is very sensitive to the injection of information that favour likely correct matches. We identify a wider challenge in crosslinking data analysis tools with maintaining the decoy-false positive relationship, and exemplify this with the filtering of results based on the information of which proteins can be observed as having reacted with the crosslinker. Without correcting for this problem, we could identify an average of 260 interspecies protein-protein interactions in 16 analyses, "solidly" suggesting groundbreaking biological connections that dont actually exist. We also show how data analysis procedures can be tested and modified to rescue the decoy-false positive relationship. The importance of this relationship for reliably identifying protein-protein interactions cannot be overstated.

bioinformatics↗

bioRxiv2023

Control of iron homeostasis by a regulatory protein-protein interaction in Bacillus subtilis: The FurA (YlaN) acts as an antirepressor to the ferric uptake regulator Fur

Iron is essential for most organisms. However, two problems are associated with the use of iron for aerobically growing organisms: (i) its accumulation leads to the formation of toxic reactive oxygen species and (ii) it is present mainly as the highly insoluble ferric iron which makes the access to iron difficult. As a consequence, a tight regulation of iron homeostasis is required. This regulation is achieved in many bacteria by the ferric uptake repressor Fur. The way how the activity of Fur is controlled, has so far remained elusive. Here, we have identified the Fur antirepressor FurA (previously YlaN) in the model bacterium Bacillus subtilis and describe its function to release Fur from the DNA under conditions of iron limitation. The FurA protein physically interacts with Fur, and this interaction prevents Fur from binding to its target sites due to a complete re-orientation of the protein. Both in vivo and in vitro experiments using a reporter fusion and Fur-DNA binding assays, respectively, demonstrate that the Fur-FurA interaction prevents Fur from binding DNA and thus from repressing the genes required for iron uptake. Accordingly, the lack of FurA results in the inability of the cell to express the genes for iron uptake under iron-limiting conditions. This explains why the furA gene was identified as being essential under standard growth conditions in B. subtilis. Phylogenetic analysis suggests that the control of Fur activity by the antirepressor FurA is confined to, but very widespread in bacteria of the class Bacilli. IMPORTANCEIron is essential for most bacteria since it is required for many redox reactions. Under aerobic conditions, iron is both essential and toxic due to radical formation. Thus, iron homeostasis must be faithfully controlled. The transcription factor Fur is responsible for this regulation in many bacteria; however, the control of Fur activity has remained open. Here we describe the FurA protein, a so far unknown protein which acts as an antirepressor to Fur in Bacillus subtilis. This mechanism seems to be widespread in B. subtilis and several important pathogens and might be a promising target for drug development.

microbiology↗

bioRxiv2023

The sterol transporter STARD3 transports sphingosine at ER-lysosome contact sites

Sphingolipids are important structural components of membranes. Additionally, simple sphingolipids such as sphingosine are highly bioactive and participate in complex subcellular signaling. Sphingolipid deregulation is associated with many severe diseases including diabetes, Parkinsons and cancer. Here, we focus on how sphingosine, generated from sphingolipid catabolism in late endosomes/lysosomes, is reintegrated into the biosynthetic machinery at the endoplasmic reticulum (ER). We characterized the sterol transporter STARD3 as a sphingosine transporter acting at lysosome-ER contact sites. Experiments featuring crosslinkable sphingosine probes, supported by unbiased molecular dynamics simulations, exposed how sphingosine binds to the lipid-binding domain of STARD3. Following the metabolic fate of pre-localized lysosomal sphingosine showed the importance of STARD3 and its actions at contact sites for the integration of sphingosine into ceramide in a cellular context. Our findings provide the first example of inter-organellar sphingosine transfer and pave the way for a better understanding of sphingolipid - sterol co-regulation.

cell biology↗

bioRxiv2023

Direct recruitment of Mis18 to interphase spindle poles promotes CENP-A chromatin assembly

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB LInker of Nucleoskeleton and Cytoskeleton (LINC) complex component Sad1. Mis18 recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1, but is independent of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

cell biology↗

bioRxiv2023

Modelling protein complexes with crosslinking mass spectrometry and deep learning

Scarcity of structural and evolutionary information on protein complexes poses a challenge to deep learning-based structure modelling. We integrated experimental distance restraints obtained by crosslinking mass spectrometry (MS) into AlphaFold-Multimer, by extending AlphaLink to protein complexes. Integrating crosslinking MS data substantially improves modelling performance on challenging targets, by helping to identify interfaces, focusing sampling, and improving model selection. This extends to single crosslinks from whole-cell crosslinking MS, opening the possibility of whole-cell structural investigations driven by experimental data. We demonstrate this by revealing the molecular basis of iron homeostasis in Bacillus subtilis.

bioinformatics↗

bioRxiv2023

"Cold" Orthogonal Translation: Psychrophilic Pyrrolysyl-tRNA Synthetase as Efficient Tool for Expanding the Genetic Code

Using orthogonal translation systems (OTSs) is one of the most efficient strategies for producing unnatural proteins through the incorporation of non-canonical amino acids (ncAAs) into the genetic code. Traditionally, efforts to expand substrate specificity start with a (hyper-)stable enzyme capable of withstanding the structural changes induced by necessary mutations. In contrast, we propose a radically different approach for the PylRS system: by starting with enzymes that evolved to cope with instability in order to adapt to cold conditions, potentially offering greater resilience to mutational changes. By finding and further engineering a psychrophilic ("cold") OTS from Methanococcoides burtonii, we developed an alternative to the widely used mesophilic and thermophilic systems. This novel OTS demonstrated exceptional in vivo efficiency for a broad range of substrates, even at very low ncAA concentrations and low cultivation temperatures. The general versatility of the PylRS system across a wide range of applicable host organisms suggests that the Cold-OTS has the potential to also improve protein yields in these hosts and help to drive the transformation of the expanded genetic code from an academic pursuit into a high-value, chemistry-driven biotechnology.

synthetic biology↗

bioRxiv2023

Ki-67 is necessary during DNA replication for forks protection and genome stability

BackgroundThe proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years but investigations on its biological functions have lagged. Recently, Ki-67 was shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, a role for Ki-67 in regulating cell cycle progression has never been reported. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein coupled to its abundance and fluctuation during the cell cycle. ResultsHere we have used an auxin-inducible degron tag (AID) to achieve a rapid and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allowed us to show for the first time that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependant on HUWE1 ligase. This leads to replication and sister chromatids cohesion defects, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. ConclusionsWe have unveiled a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.

cell biology↗

bioRxiv2023

Cleavable crosslinkers redefined by novel MS3-trigger algorithm

Crosslinking MS is currently transitioning from a routine tool in structural biology to enabling structural systems biology. MS-cleavable crosslinkers could substantially reduce the associated search space expansion by allowing an MS3-based approach for identifying crosslinked peptides. However, MS2-based approaches currently outperform approaches utilising MS3. We show here that MS3-trigger sensitivity and specificity were hampered algorithmically. Our four-step MS3-trigger algorithm greatly outperformed currently employed methods and comes close to reaching the theoretical limit.

bioinformatics↗

bioRxiv2022

AI-guided cryo-EM probes a thermophilic cell-free system with succinyl-coA manufacturing capability

Cell-free systems display tremendous potential for biotechnological applications, complementing in vitro reconstituted enzymatic processes and traditional expression systems. However, they often represent "black boxes" without much insight into their components. Here, we characterize a thermophilic cell-free system that produces succinyl-CoA and discern its intrinsic, non-stochastic organization. By employing biochemical, biophysical, and bioinformatic methods we resolve its molecular composition, 3D architecture and molecular function at atomic resolution. We further report the high-resolution cryo-EM structure of the reactions main component, the oxoglutarate dehydrogenase complex core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-communication between subunits, and the presence of a flexible subunit, the E3BPo connecting E2o and E3. This multi-scale analysis of a cell-free system provides a blueprint for structure-function studies of complex mixtures of biotechnological value.

biochemistry↗

bioRxiv2022

DomainRank: Improving Biological Data Sets With Domain Knowledge and Google's PageRank

MotivationThe quality of biological data crucially affects progress in science. This quality can be improved with better measurement devices, more sophisticated experimental designs, or repetitious measurements. Each of these options is associated with substantial costs. We present a simple computational tool as an alternative. This algorithmic tool, called DomainRank, leverages simple domain knowledge and overlapping information content in biological network data to improve measurement quality at a negligible cost. Following the simple computational template of Domain-Rank, researchers can boost the confidence of their own data with little effort. ResultsWe demonstrate the performance of DomainRank in three test cases: DomainRank finds 14.9% more interactions in quantitative proteomics experiments, improves the precision of predicted residue-residue contacts from co-evolutionary data by up to 11.6% (averaged over 882 proteins), and identifies 89.2% more cross-links in photo-crosslinking/mass spectrometry (photo-CLMS) experiments. Although our proposed template is specialized on biological network data, we view this approach as an universal computational tool for data improvement that could be routinely applied in many disciplines. AvailabilityAn implementation of DomainRank is freely available: https://github.com/Rappsilber-Laboratory/pagerank-refine

bioinformatics↗

bioRxiv2022

DAXX adds a de novo H3.3K9me3 deposition pathway to the histone chaperone network

A multitude of histone chaperones are required to protect histones after their biosynthesis until DNA deposition. They cooperate through the formation of co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using explorative interactomics approaches, we characterize the organization of the histone H3-H4 chaperones network and define the interplay between histone chaperone systems. We identify and validate several novel histone dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX acts separately from the rest of the network, recruiting heterochromatin factors and promoting lysine 9 tri-methylation of new histone H3.3 prior to deposition onto DNA. With its functionality, DAXX provides a molecular mechanism for de novo heterochromatin assembly. Collectively, our findings provide a new framework for understanding how cells orchestrate histone supply and comply with chromatin dynamics throughout the cell cycle.

molecular biology↗

bioRxiv2022

Protein Complexes in Bacillus subtilis by AI-assisted structural proteomics

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enables a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry, cofractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interface, and is applicable to genetically intractable organisms, including pathogenic bacteria.

molecular biology↗

bioRxiv2022

Specific recognition and ubiquitination of slow-moving ribosomes by human CCR4-NOT

Eukaryotic messenger RNA (mRNA) decay is generally initiated by removal of the 3 polyadenosine (poly(A)) tail by the CCR4-NOT complex. Yeast Ccr4-Not binds and ubiquitinates ribosomes stalled on mRNAs with sub-optimal codons to trigger deadenylation and decay of the associated transcript. However, the mammalian ortholog of the E3 ubiquitin ligase subunit, CNOT4, is not a constitutive component of human CCR4-NOT. It therefore remains unclear how the mammalian deadenylation machinery targets stalled ribosomes. Here, we reconstitute translational stalling on non-optimal codons. We find that human CCR4-NOT recognizes translating mammalian ribosomes and is required for stable CNOT4 association. Our cryoEM structure reveals that the CNOT3 subunit detects slow translation and locks the L1 stalk of the ribosome in an open conformation to impede further elongation. Using crosslinking mass spectrometry, we show that CNOT4 and CNOT11 also bind in the vicinity of the E site. Overall, our work defines how CCR4-NOT enforces ribosomal stalling in response to low codon optimality.

molecular biology↗