Biology subjects

Hurt, E.

Publications and source records attributed to Hurt, E..

9 recordsLinked to original sources

bioRxiv2026

RNA helicase Drs1 gates 25S rRNA domain III incorporation during early nucleolar pre-60S maturation

Assembly of eukaryotic large ribosomal subunits (LSU) requires coordinated structural and compositional transitions within pre-60S particles, yet the underlying mechanisms remain poorly understood. Here, we show that the DEAD-box helicase Drs1 promotes early maturation across distinct regions of the pre-60S particle. Loss of Drs1 function causes accumulation of co-transcriptional intermediates retaining SSU processome components, indicating that Drs1 promotes timely separation of nascent LSU precursors from the small-subunit assembly pathway. Cryo-EM analyses reveal both a redistribution toward early nucleolar maturation states upon loss of Drs1, including Nsa1-deficient intermediates, and a confinement of Drs1-associated particles to states preceding stable incorporation of 25S rRNA domain III. Drs1 directly engages Erb1 through its unstructured N-terminal extension, promoting stable assembly of the Nop7-Erb1-Ytm1 module associated with domain III maturation. CRAC analysis localizes Drs1 to spatially clustered sites spanning the 5.8S and 25S rRNAs, encompassing domains I-IV. Together, these findings support a model in which Drs1 couples stabilization of assembly-factors with pre-rRNA remodeling across the pre-60S particle, thereby driving ordered early LSU maturation and the timed integration of domain III.

biochemistry↗

bioRxiv2026

A bifunctional H/ACA snoRNP mediates both pseudouridylation and rRNA scaffolding during ribosome assembly

The early steps of eukaryotic large ribosomal subunit assembly remain poorly understood due to the structural flexibility of pre-60S intermediates, whose rRNA is extensively modified by small nucleolar RNPs (snoRNPs). Some snoRNPs, however, lack any modification function and instead scaffold ribosome assembly through largely unknown mechanisms. Here, we show that the H/ACA snoRNP snR37 integrates both modifying and scaffolding roles. Biochemical and structural analyses reveal a canonical H/ACA core that pseudouridylates a conserved uridine in the A site of the peptidyl transferase center, the catalytic heart of the 60S subunit. Additional RNA helices recruit non-core proteins, the Upa1-Upa2 heterodimer and Rbp95, which mediate stable snR37 association with pre-60S complexes. These proteins cooperate with the Npa1 rRNA chaperone complex to link four rRNA domains, thereby structurally organizing early pre-60S intermediates and promoting proper formation of the PTC. This dual organization establishes a paradigm for snoRNPs combining rRNA modification and scaffolding functions.

biochemistry↗

bioRxiv2024

H/ACA snoRNP guides ribosomal RNA subdomain folding in a satellite particle before joining the core 90S pre-ribosome

During synthesis, modification and maturation of the ribosomal RNA, correct subdomain folding without additional guidance poses a major challenge. An essential H/ACA snoRNP, snR30, was observed to be outsourced from the core of the 90S, the earliest known pre-ribosome, to form a "satellite particle". This particle interacts with the so-called 18S rRNA platform subdomain, guiding its independent folding through localized structural interactions. Once this subdomain achieves its proper conformation, it is integrated into the core pre-ribosome, contributing to the overall maturation of the 18S rRNA. By temporarily segregating the folding process within a specialized satellite particle, the correct assembly of individual components is facilitated, thereby reducing the risk of errors during the complex process of ribosome assembly. In a broader sense, molecular outsourcing in cellular processes, where transient subcomplexes handle specific tasks before rejoining the main assembly line, can enhance efficiency and accuracy of complex molecular processes like ribosome biogenesis.

biochemistry↗

bioRxiv2024

Evaluating computational approaches for comparison of protein expression across cancer indications

BackgroundThe National Cancer Institutes Clinical Proteomic Tumor Analysis Consortium (CPTAC) recently generated harmonized genomic, transcriptomic, proteomic, and clinical data for over 1,000 tumors across 10 cohorts to facilitate pan-cancer discovery research. However, protein expression comparison across CPTAC cohorts remains challenging due to non-uniform missing data and varying protein expression distribution patterns across tumor types. Here, we present our efforts to evaluate various missing data handling and normalization strategies to create a normalized pan-cancer protein expression dataset. ResultsFirst, we developed a novel algorithm to select robustly expressed proteins in tumors within any CPTAC cohort. Second, we applied a cohort hybrid imputation approach to protein abundance values from FragPipe within each cohort based on protein expression distribution patterns. Third, we calculated intensity-based absolute quantification using protein abundance values and applied both global and smooth quantile normalization methods. Our results indicate that global quantile normalization ensured identical distribution across cohorts for both tumor and normal tissues, while smooth quantile normalization preserved distribution differences between biological conditions. We assessed our method by comparing differential protein expression analysis results with and without normalization. Additionally, we examined the ranks of protein expression in the normalized CPTAC dataset for selected proteins with high protein-to-RNA expression correlation across CPTAC cohorts. We then compared these protein expression ranks with their RNA expression ranks across corresponding cohorts in The Cancer Genome Atlas (TCGA). Differential protein expression analysis revealed a high level of agreement in the fold change of tumor versus normal tissue within cohorts before and after normalization. Furthermore, our results indicate that global quantile normalization resulted in the highest cohort rank correlation between CPTAC and TCGA for selected proteins. ConclusionsIn summary, our thorough analysis demonstrates that global quantile normalization surpasses both smooth quantile normalization and no normalization, as evidenced by its higher rank correlation across cancer cohorts between CPTAC and TCGA for selected proteins. The findings suggest that combining cohort hybrid imputation with global quantile normalization is an effective method for creating a normalized CPTAC pan-cancer protein dataset, which can facilitate the study of protein expression across different cancer types.

bioinformatics↗

bioRxiv2023

Pan-cancer Proteomics Analysis to Identify Tumor-Enriched and Highly Expressed Cell Surface Antigens as Potential Targets for Cancer Therapeutics

The National Cancer Institutes Clinical Proteomic Tumor Analysis Consortium (CPTAC) provides unique opportunities for cancer target discovery using protein expression. Proteomics data from CPTAC tumor types have been primarily generated using a multiplex tandem mass tag (TMT) approach, which is designed to provide protein quantification relative to reference samples. However, relative protein expression data is suboptimal for prioritization of targets within a tissue type, which requires additional reprocessing of the original proteomics data to derive absolute quantitation estimation. We evaluated the feasibility of using differential protein analysis coupled with intensity-based absolute quantification (iBAQ) to identify tumor-enriched and highly expressed cell surface antigens, employing tandem mass tag (TMT) proteomics data from CPTAC. Absolute quantification derived from TMT proteomics data was highly correlated with that of label-free proteomics data from the CPTAC colon adenocarcinoma cohort, which contains proteomics data measured by both approaches. We validated the TMT-iBAQ approach by comparing the iBAQ value to the receptor density value of HER2 and TROP2 measured by flow cytometry in about 30 selected breast and lung cancer cell lines from the Cancer Cell Line Encyclopedia. Collections of these tumor-enriched and highly expressed cell surface antigens could serve as a valuable resource for the development of cancer therapeutics, including antibody-drug conjugates and immunotherapeutic agents.

bioinformatics↗

bioRxiv2022

Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodelling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

biochemistry↗

bioRxiv2022

New motifs in Ndc1 mediating interaction with the Nup84 complex and nuclear membranes

The nuclear pore complex (NPC) embedded in the double nuclear membrane is built from ~30 different nucleoporins (Nups) in multiple copies, of which a few are integral nuclear membrane proteins. One of these transmembrane Nups is Ndc1, which is thought to play a role in interphase NPC assembly at the fused inner and outer nuclear membrane. In this study, we discovered a direct interaction of Ndc1s transmembrane domain with Nup120 and Nup133, members of the Y-complex that coats the nuclear pore membrane. In addition, we identified a so far unrecognized amphipathic helix (AH) in the C-terminal domain of Ndc1, which can bind to high curvature liposomes. When overexpressed in yeast this amphipathic motif is toxic and dramatically alters the intracellular membrane organization. Further genetic investigations revealed that Ndc1-AH functionally interacts with related motifs in the C-terminus of Nups Nup53 and Nup59, known to serve in nuclear pore membrane binding and link between NPC modules. This relationship could explain why the essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane biogenesis dependent on a balanced ratio between amphipathic motifs in diverse nucleoporins is essential for interphase NPC biogenesis.

cell biology↗

bioRxiv2022

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

Folding of nascent chains emerging from the ribosome is a challenge in cellular protein homeostasis, which in eukaryotes is met by an Hsp70 chaperone triad directly binding at the ribosomal tunnel exit. The conserved ribosome-associated complex (RAC) consists of the non-canonical Hsp70 Ssz1 and the J-domain protein Zuotin (Zuo1), which in fungi acts together with the canonical Hsp70 protein Ssb. Here, we determined high-resolution cryo-electron microscopy structures of RAC bound to the 80S ribosome. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. The heterodimer is held together by a tight interaction between the Ssz1 substrate-binding domain (SBD) and the N-terminus of Zuo1, with additional contacts between the Ssz1 nucleotide-binding domain (NBD) and the Zuo1 J- and ZHD domains that form a rigid unit. The Zuo1 HPD-motif conserved in J-proteins is masked by the Ssz1 NBD, different from the canonical Hsp70 J-protein contact, however, allowing to position Ssb for activation by Zuo1. Our data provide the basis for understanding how RAC cooperates with Ssb at the ribosome in dynamic nascent chain interaction and protein folding.

biochemistry↗

bioRxiv2019

Global Transcriptome Characterization and Assembly of Thermophilic Ascomycete Chaetomium thermophilum

A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome Chaetomium thermophilum has been reported previously, but it is limited to open reading frames (ORFs) of genes and contains only a few noncoding transcripts. In this study, we identified and annotated by deep RNA sequencing full-length transcripts of C.thermophilum. We annotated 7044 coding genes and a large number of noncoding genes (n=4567). Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help understanding the gene regulatory landscape in C.thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species. The R packages for gene and GO annotation database can be found under https://www.bzh.uni-heidelberg.de/brunner/Chaetomium_thermophilum.

bioinformatics↗