Biology subjects

Klaholz, B. P.

Publications and source records attributed to Klaholz, B. P..

6 recordsLinked to original sources

bioRxiv2026

A ribosomal pseudouridine drives an oncogenic translation program

Dynamic ribosome heterogeneity has emerged as a potential layer of translational regulation, but whether variation in ribosomal RNA (rRNA) modification stoichiometry can selectively control the translation of mRNAs with shared encoded features remains unclear. Here we show that SNORA81-guided pseudouridylation at U4636 controls a selective translational program in ovarian cancer cells. U4636 is partially pseudouridylated and lies within 28S rRNA near eL31 and the peptide exit tunnel. Depletion of SNORA81 reduces pseudouridylation at U4636 without broadly perturbing rRNA processing or bulk translation. Instead, it remodels codon-specific and nascent-peptide-associated pausing and the polysome association of SNORA81-dependent translationally altered mRNAs. These mRNAs display characteristic GC content and amino-acid composition, are enriched in cancer-related functions and show corresponding changes in protein levels. Our findings support a model in which a change in the abundance of a single snoRNA propagates from rRNA-modification stoichiometry through ribosome dynamics and selective mRNA translation to protein output and cancer cell proliferation. We term this multilevel amplification cascade the "sno-balling" effect.

cancer biology↗

bioRxiv2025

EXPLICIT CORRECTION OF SEVERELY NON-UNIFORM DISTRIBUTIONS OF CRYO-EM VIEWS

The quality of three-dimensional macromolecular image reconstructions by cryo electron microscopy strongly depends on the number and quality of respective two-dimensional projections and on their angular distribution in space. Distributions with one or a few strongly preferred particle orientations may result in maps deformed in certain directions. A simple removal of overrepresented views may improve the quality of the reconstructed maps when the level of noise in the two-dimensional projections is low and the dataset-size can afford this removal but is counterproductive or insufficient otherwise. Giving complementarily an increased weight to underrepresented views, or taking multiple copies of them during the reconstruction, may improve the results, naturally, depending on how much the views distribution is non-uniform. This work describes the results of three-dimensional reconstructions using an explicit correction of the number of over- and underrepresented projections for non-uniformly distributed sets. SynopsisAn explicit numerical leveling of non-uniformly distributed sets of 2D projections with the program VUE improves the 3D reconstructions and illustrate sources of image distortions.

molecular biology↗

bioRxiv2023

Structure of the human 80S ribosome at 1.9 A resolution - the molecular role of chemical modifications and ions in RNA

The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. We present the 1.9 [A] resolution cryo-EM structure of the 80S human ribosome resolving numerous new rRNA modifications and functionally important ions such as Zn2+, K+ and Mg2+ including their associated individual water molecules. 2-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry resulting in a complete investigation of the > 230 sites many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerisation turns out to be a key mechanism for U-A base pair stabilisation in RNA in general. The structural environment of chemical modifications & ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy context and human diseases.

biochemistry↗

bioRxiv2021

A spectral demixing method for high-precision multi-color localization microscopy

Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. So far, demixing algorithms give suboptimal results in terms of localization precision and correction of chromatic aberrations. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision & drift correction, compensation of chromatic aberrations, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic aberrations at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.

biophysics↗

bioRxiv2018

New tools for the analysis and validation of Cryo-EM maps and atomic models

Recent advances in the field of electron cryo-microscopy (cryo-EM) have resulted in a rapidly increasing number of atomic models of bio-macromolecules solved using this technique and deposited in the Protein Data Bank and the Electron Microscopy Data Bank. Similar to macromolecular crystallography, validation tools for these models and maps are required. While some of these validation tools may be borrowed from crystallography, new methods specifically for cryo-EM validation are required. We discuss new computational methods and tools implemented in Phenix, including d99 to estimate resolution, phenix.auto_sharpen to improve maps, and phenix.mtriage to analyze cryo-EM maps. We suggest that cryo-EM half-maps and masks are deposited to facilitate evaluation and validation of cryo-EM derived atomic models and maps. We also present the application of these tools to deposited cryo-EM atomic models and maps.

bioinformatics↗

bioRxiv2018

Tetracyclines Modify Translation By Targeting Key Human rRNA Substructures.

Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

cell biology↗