Biology subjects

Klaholz, B.

Publications and source records attributed to Klaholz, B..

8 recordsLinked to original sources

bioRxiv2025

Translation initiation by the Kozak mRNA sequence is based on a conformational readout on the ribosome

The recognition mechanism of Kozak mRNA, typically comprising purines in the - 3 and +4 positions flanking the AUG start codon, has remained enigmatic for decades. To address this fundamental function in eukaryotes during translation initiation, we analysed the cryo-EM structures of human 48S preinitiation complexes with mRNA point mutations. They reveal a fan-like intercalation of the pre-codon triplet into the 18S ribosomal RNA (rRNA), while the -3 purine as opposed to the pyrimidine in the non-Kozak context favours stabilization of the ternary complex between initiation factor eIF2 and initiator tRNA. Specificity towards the +4 purine is achieved beyond a single residue recognition by mutual conformational adaptations of eIF1A, mRNA and rRNA and involves the insertion of a reading head in which decoding residue A1825 (rRNA) stacks with the A-site codon to stabilize the fully accommodated state. Hence, instead of relying on base pairing as in bacteria, the specific recognition of the Kozak sequence on eukaryotic ribosomes is based on an induced-fit mechanism that triggers a conformational readout of the mRNA.

biochemistry↗

bioRxiv2025

Extended Shine-Dalgarno motifs govern translation initiation in Staphylococcus aureus

Regulation of translation initiation is central to bacterial adaptation, but species-specific mechanisms remain poorly understood. We present high-resolution mapping of translation start sites in S. aureus, revealing distinct features of initiation alongside numerous unannotated small ORFs. Our analysis, combined with cryo-EM of a native mRNA-ribosome complex, shows that S. aureus relies on extended, start codon proximal Shine-Dalgarno (SD) interactions, creating specificity against phylogenetically distant bacteria. Several natural S. aureus initiation sites are not correctly decoded by E. coli ribosomes. We identify new and conserved non-canonical start codons, whose regulatory initiation sites contain these characteristic extended SD sequence motifs. Finally, we characterize a striking example of uORF-mediated translational control in S. aureus, demonstrating that translation of a novel small leader peptide modulates expression of a key biofilm regulator. The described mechanism involves codon rarity, ribosome pausing and arginine availability, linking nutrient sensing to biofilm formation in this major human pathogen. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/660503v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ea5094org.highwire.dtl.DTLVardef@505be5org.highwire.dtl.DTLVardef@1c14ad6org.highwire.dtl.DTLVardef@118549c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

Annulate Lamellae biogenesis is essential for nuclear pore function

Nuclear pore complexes (NPCs) enable nucleocytoplasmic transport. While NPCs primarily localize to the nuclear envelope (NE), they also appear in cytoplasmic endoplasmic reticulum (ER) membranes called annulate lamellae (AL). Though discovered in the mid-20th century, ALs function and biogenesis remain unclear. Previously considered exclusive to embryonic and malignant cells, we find AL in somatic mammalian cells. Under normal conditions, AL store pre-assembled NPCs (AL-NPCs) that integrate into the NE during G1 to support nuclear expansion. Upon pathological stimuli, AL transfer to the NE is impaired, leading to their cytoplasmic accumulation. RanBP2 (Nup358) is essential for AL biogenesis, with its phenylalanine-glycine (FG) repeats promoting AL-NPC scaffold oligomerization. ER-associated Climp63 (CKAP4) directs AL-NPCs to ER sheets and the NE. This AL-driven nuclear pore formation is complementary to the canonical routes, constituting a distinct NPC assembly pathway. Our work uncovers the biogenesis mechanism of AL and the nuclear function of this key cellular organelle.

cell biology↗

bioRxiv2023

RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in Staphylococcus aureus

rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analysis of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general as well as species-specific roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in E. coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.

molecular biology↗

bioRxiv2023

UBAP2L drives scaffold assembly of nuclear pore complexes at the intact nuclear envelope

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a direct role of ubiquitin-associated protein 2-like (UBAP2L) in the biogenesis of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and drives the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L facilitates the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact nuclear envelope. TeaserLiao et al. show how UBAP2L drives the assembly of the scaffold elements into symmetrical and functional NPCs at the nuclear envelope in human cells.

cell biology↗

bioRxiv2020

METTL5, an 18S rRNA-specific m6A methyltransferase, modulates expression of stress response genes

RNA N6-methyladenosine (m6A) modification is present in different RNA molecules, including protein-coding mRNAs and non-coding RNAs such as ribosomal RNAs (rRNAs). Previous studies identified m6A in both the 18S and 28S rRNAs, but the roles of these methylation events are poorly understood due to the lack of knowledge of the responsible methyltransferases. Here, we report that mammalian METTL5, a member of a highly conserved methyltransferase family, specifically methylates adenosine 1832 (A1832) in the 18S rRNA in vivo and in vitro. We identify TRMT112 as a near stoichiometric partner of METTL5 important for the enzymatic activity of METTL5. By mapping the positions of translating ribosomes (Ribo-seq), we found translation of multiple stress response-related mRNAs, including Atf4 mRNA, is selectively reduced in the Mettl5 knockout (KO) mouse B16 melanoma cells. Atf4 is a key transcription factor that mediates the Integrated Stress Response (ISR), as exemplified by the Endoplasmic Reticulum (ER) stress. Consistently, transcription of ISR effector genes is reduced in Mettl5 KO cells during ER stress, suggesting a compromised ISR. Our findings reveal a new mechanism that regulates expression of stress response genes and suggest that chemical modifications of ribosomal RNAs may play a key role in selectively impacting translation and possibly ISR.

biochemistry↗

bioRxiv2019

Structural basis of nanobody-recognition of grapevine fanleaf virus and of virus resistance loss

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently and shown to be highly effective in plants including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb23 complex which provides the basis for the molecular recognition by the nanobody. The structure reveals a composite binding site bridging over 3 domains of the capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.\n\nSignificanceGrapevine fanleaf virus (GFLV) is a picorna-like plant virus that severely impacts vineyards worldwide. While Nanobodies (Nb) confer resistance to GFLV in plants the underlying molecular mechanism of action is unknown. Here we present the high-resolution cryo-EM structure of the GFLV-Nb complex. It uncovers the conformational epitope on the capsid surface which is a composite binding site into which the antigen loop is accommodated through an induced fit mechanism. Furthermore, we describe several resistance-breaking isolates of GFLV with reduced Nb binding capacity. Those that carry a C-terminal extension also fail to be transmitted by nematodes. Together, these data provide structure-function insights into the Nb-GFLV recognition and the molecular mechanism leading to loss of resistance.

biochemistry↗

bioRxiv2017

3D clustering analysis of super-resolution microscopy data by 3D Voronoi tessellations

Single-molecule localization microscopy (SMLM) can play an important role in integrated structural biology approaches for example at the interface of cryo electron microscopy (cryo-EM), X-ray crystallography, NMR and fluorescence imaging to identify, localize and determine the 3D structure of cellular structures. While many tools exist for the 3D analysis and visualisation of crystal or cryo-EM structures little exists for 3D SMLM data which can provide fascinating insights but are particularly challenging to analyze in three dimensions especially in a dense cellular context. We developed 3DClusterViSu, a method based on 3D Voronoi tessellations that allows local density estimation, segmentation & quantification of 3D SMLM data and visualization of protein clusters within a 3D tool. We show its robust performance on microtubules and histone proteins H2B and CENP-A with distinct spatial distributions. 3DClusterViSu will favor multi-scale and multi-resolution synergies to allow integrating molecular and cellular levels in the analysis of macromolecular complexes.

bioinformatics↗