Biology subjects

Steckelberg, A.-L.

Publications and source records attributed to Steckelberg, A.-L..

9 recordsLinked to original sources

bioRxiv2025

Template-based RNA structure prediction advanced through a blind code competition

Automatically predicting RNA 3D structure from sequence remains an unsolved challenge in biology and biotechnology. Here, we describe a Kaggle code competition engaging over 1700 teams and 43 previously unreleased structures to tackle this challenge. The top three submitted algorithms achieved scores within statistical error of the winners of the recent CASP16 competition. Unexpectedly, the top Kaggle strategy involved a pipeline for discovering 3D templates, without the use of deep learning. We integrated this template-modeling pipeline and other Kaggle strategies to develop a single model RNAPro that retrospectively outperformed individual Kaggle models on the same test set. These results suggest a growing importance of template-based modeling in RNA structure prediction.

biophysics↗

bioRxiv2025

Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends or a base forms a non-standard hydrogen bond. These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

biophysics↗

bioRxiv2025

Ku suppresses RNA-mediated innate immune responses in human cells to accommodate primate-specific Alu expansion.

Ku70 and Ku80 form Ku, a ring-shaped protein that initiates the non-homologous end-joining (NHEJ) DNA repair pathway.1 Specifically, Ku binds to double-stranded DNA (dsDNA) ends and recruits other NHEJ factors (e.g., DNA-PKcs and LIG4). While Ku binds to double-stranded RNA (dsRNA)2 and traps mutated-DNA-PKcs on ribosomal RNA in vivo,3,4 the physiological significance of Ku-dsRNA interactions in otherwise wild-type cells remains elusive. Intriguingly, while dispensable for murine development,5,6 Ku is essential in human cells.7 Despite similar genome sizes, human cells express [~]100-fold more Ku than mouse cells, implying functions beyond NHEJ, possibly through a dose-sensitive interaction with dsRNA, which is [~]100 times weaker than with dsDNA.2,8 While investigating the essentiality of Ku in human cells, we found that depletion of Ku - unlike LIG4 - induces profound interferon (IFN) and NF-kB responses reliant on the dsRNA-sensor MDA5/RIG-I and adaptor MAVS. Prolonged Ku-degradation also activates other dsRNA-sensors, e.g. PKR that suppresses protein translation, and OAS/RNaseL that cleaves rRNAs and eventually induces growth arrest and cell death. MAVS, RIG-I, or MDA5 knockouts suppressed IFN signaling and, together with PKR knockouts, partially rescued Ku-depleted human cells. Ku-irCLIP analyses revealed that Ku binds to diverse dsRNA, predominantly stem-loops in primate-specific Alu elements9 at anti-sense orientation in introns and 3-UTRs. Ku expression rose sharply in higher primates tightly correlating with Alu-expansion (r = 0.94/0.95). Together, our study identified a vital role of Ku in accommodating Alu-expansion in primates by mitigating a dsRNA-induced innate immune response, explaining the rise of Ku levels and its essentiality in human cells.

genetics↗

bioRxiv2024

The pseudoknot structure of a viral RNA reveals a conserved mechanism for programmed exoribonuclease resistance

Exoribonuclease-resistant RNAs (xrRNAs) are viral RNA structures that block degradation by cellular 5'-3' exoribonucleases to produce subgenomic viral RNAs during infection. Initially discovered in flaviviruses, xrRNAs have since been identified in wide range of RNA viruses, including those that infect plants. High sequence variability among viral xrRNAs raises questions about the shared molecular features that characterize this functional RNA class. Here, we present the first structure of a plant-virus xrRNA in its active exoribonuclease-resistant conformation. The xrRNA forms a 9 base pair pseudoknot that creates a knot-like topology similar to that of flavivirus xrRNAs, despite lacking sequence similarity. Biophysical assays confirm a compact pseudoknot structure in solution, and functional studies validate its relevance both in vitro and during infection. Our study reveals how viral RNAs achieve a common functional outcome through highly divergent sequences and identifies the knot-like topology as a defining feature of xrRNAs.

biochemistry↗

bioRxiv2023

An evolutionarily conserved strategy for ribosome binding and inhibition by β-coronavirus non-structural protein 1

An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Nsp1, which suppresses host gene expression and stunts antiviral signaling. SARS-CoV-2 Nsp1 binds the ribosome to inhibit translation through mRNA displacement and induces degradation of host mRNAs through an unknown mechanism. Here we show that Nsp1-dependent host shutoff is conserved in diverse coronaviruses, but only Nsp1 from {beta}-CoV inhibits translation through ribosome binding. The C-terminal domain of all {beta}-CoV Nsp1s confers high-affinity ribosome-binding despite low sequence conservation. Modeling of interactions of four Nsp1s to the ribosome identified few absolutely conserved amino acids that, together with an overall conservation in surface charge, form the {beta}-CoV Nsp1 ribosome-binding domain. Contrary to previous models, the Nsp1 ribosome-binding domain is an inefficient translation inhibitor. Instead, the Nsp1-CTD likely functions by recruiting Nsp1s N-terminal "effector" domain. Finally, we show that a viral cis-acting RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. Together, our work provides new insight into the diversity and conservation of ribosome-dependent host-shutoff functions of Nsp1, knowledge that could aide future efforts in pharmacological targeting of Nsp1 from SARS-CoV-2, but also related human-pathogenic {beta}-coronaviruses. Our study also exemplifies how comparing highly divergent Nsp1 variants can help to dissect the different modalities of this multi-functional viral protein.

biochemistry↗

bioRxiv2020

Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA for destruction. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific 3-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana Bat Virus (TABV) and solved its structure by x-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

biochemistry↗

bioRxiv2020

The crystal structure of a Polerovirus exoribonuclease-resistant RNA shows how diverse sequences are integrated into a conserved fold

Exonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5 to 3 exonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.

biochemistry↗

bioRxiv2018

Pervasiveness of exoribonuclease-resistant RNAs in plant viruses suggests new roles for these conserved RNA structures

Exoribonuclease-resistant RNAs (xrRNAs) are discrete folded RNA elements that block the processive degradation of RNA by exoribonucleases. xrRNAs found in the 3' untranslated regions (UTRs) of animal-infecting flaviviruses and in all three members of the plant-infecting Dianthovirus adopt a complex ring-like fold that blocks the exoribonuclease; this ability gives rise to viral non-coding subgenomic RNAs. The degree to which these folded RNA elements exist in other viruses and in diverse contexts has been unclear. Using computational tools and biochemical assays, we discovered that xrRNA elements are widely found in viruses belonging to the Tombusviridae and Luteoviridae families of plant-infecting RNA viruses, demonstrating their importance and widespread utility. Unexpectedly, many xrRNAs are located in intergenic regions rather than in the 3UTR and some are associated with the 5' ends of subgenomic RNAs with protein-coding potential, suggesting that xrRNAs with similar scaffolds are involved in the maturation or maintenance of diverse subgenomic RNAs, not just the ones generated from the 3'UTR.

microbiology↗

bioRxiv2018

A folded viral noncoding RNA blocks host cell exoribonucleases through programmed remodeling of RNA structure

Folded RNA elements that block processive 5'[->]3' cellular exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the dianthovirus xrRNA can use co-degradational remodeling, exploiting the exoribonucleases degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

biochemistry↗