Biology subjects

Klinge, S.

Publications and source records attributed to Klinge, S..

11 recordsLinked to original sources

bioRxiv2026

Mechanism for the initiation of co-transcriptional pre-60S assembly

Eukaryotic ribosomal large subunit (60S) assembly requires an internal transcribed spacer 2 (ITS2) to license both nucleolar and nuclear pre-60S assembly intermediates. The underlying molecular mechanisms responsible for nucleation of pre-60S assembly, quality control, and installation of ITS2 during co-transcriptional stages remain unknown. Here we report the earliest co-transcriptional assembly intermediates of the eukaryotic 60S subunits. Together with biochemical assays, our data reveal the architecture of co-transcriptional pre-60S assembly initiation and progression, as well as the molecular logic of an assembly checkpoint. This study highlights an evolutionary solution by which complex RNA folding processes can be parallelized and integrated via biological AND-gating.

biochemistry↗

bioRxiv2025

Helicase-mediated mechanism of SSU processome maturation and disassembly

Eukaryotic ribosomal small subunit (SSU) assembly requires the SSU processome, a nucleolar precursor containing the RNA chaperone U3 snoRNA. The underlying molecular mechanisms of SSU processome maturation, remodeling, disassembly, RNA quality control, and the transitions between states remain elusive due to a paucity of intermediates1-3. Here we report 16 native SSU processome structures alongside genetic data, revealing how two helicases, the Mtr4-exosome and Dhr1, are controlled for accurate and unidirectional ribosome biogenesis. Our data show how irreversible pre-ribosomal RNA degradation by the redundantly tethered RNA exosome couples the transformation of the SSU processome into a pre-40S particle during which Utp14 can probe evolving surfaces, ultimately positioning and activating Dhr1 to unwind the U3 snoRNA and initiate nucleolar pre-40S release. This study highlights a paradigm for large dynamic RNA-protein complexes where irreversible RNA degradation drives compositional changes and communicates these changes to govern enzyme activity while maintaining overall quality control.

biochemistry↗

bioRxiv2024

Mapping and engineering RNA-controlled architecture of the multiphase nucleolus

Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a layered multiphase liquid-like structure and the site of ribosome biogenesis. In the nucleolus, ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps that ultimately result in formation of the ribosomal small subunit (SSU) and large subunit (LSU). However, how rRNA processing is coupled to the layered nucleolar organization is poorly understood due to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here, we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. Furthermore, by generating synthetic de novo nucleoli through an engineered rDNA plasmid system in cells, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.

cell biology↗

bioRxiv2023

Principles of human pre-60S biogenesis

During early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5-3.2 [A]. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how GTPases and ATPases couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA processing complex, the rixosome, couples large-scale RNA conformational changes to pre-rRNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation to elucidate the molecular principles of ribosome formation. One-Sentence SummaryHigh-resolution cryo-EM structures of human pre-60S particles reveal new principles of eukaryotic ribosome assembly.

biochemistry↗

bioRxiv2023

A co-transcriptional ribosome assembly checkpoint controls nascent large ribosomal subunit maturation

During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel, and together with ribosomal proteins cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.

biophysics↗

bioRxiv2022

Allosteric interactions prime androgen receptor dimerization and activation

The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.

biochemistry↗

bioRxiv2019

Numerical simulation of the viral entry into a cell driven by the receptor diffusion

This study focuses on the receptor driven endocytosis typical of viral entry into a cell. A locally increased density of receptors at the time of contact between the cell and the virus is necessary in this case. The virus is considered as a substrate with fixed receptors on its surface, while the receptors of the host cell are free to move over its membrane, allowing a local change in their concentration. In the contact zone the membrane inflects and forms an envelope around the virus. The created vesicle imports its cargo into the cell. The described process is simulated by the diffusion equation accompanied by two boundary conditions. The first boundary condition states that the conservation of binders expressed as the local rate of change of density has to be equal to the negative of the local flux divergence. The second boundary condition represents the energy balance condition with contributions due to the binding of receptors, the free energy of the membrane, its curvature and the kinetic energy due to the motion of the front. The described moving boundary problem in terms of the binder density and the velocity of the adhesion front is well posed and relies on biomechanically motivated assumptions. The problem is numerically solved by using the finite difference method, and the illustrative examples have been chosen to show the influence of the mobility of the receptors and of their initial densities on the velocity of the process.\n\nSIGNIFICANCEThe receptor driven endocytosis represents one of the most important mechanisms for the viral entry into a cell. However, the high velocities and small characteristic length scale cause many difficulties during the experimental investigation of such a process. This calls upon the application of virtual computer simulations investigating the process parameters and identifying factors inhibiting or completely ceasing the viral entry into cells. The development of methods for the optimization of the cell immunity system is aimed to as the final goal.

cell biology↗

bioRxiv2018

Assembly and early maturation of large subunit precursors

The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As pre-ribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well-characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors but how these particles emerge is still unknown. Here, we use the overexpression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II and VI of the assembling 25S rRNA.

biochemistry↗

bioRxiv2018

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here eleven new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that six of eighteen (33%) protein-coding mutations and the two (100%) 5-UTR mutations display incomplete penetrance. Three mutations were identified in 2 independent kindreds, due to a hotspot or a founder effect. Lastly, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

genetics↗

bioRxiv2017

Modular assembly of the nucleolar large subunit processome

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 [A]ngstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

biochemistry↗

bioRxiv2017

The complete structure of the small subunit processome

The small subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small subunit processome at an overall resolution of 3.8 [A], which provides an essentially complete atomic model of this assembly. In this nucleolar superstructure, 51 ribosome assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry, steric hindrance as well as protein-and RNA-mediated RNA remodeling are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

biochemistry↗