Biology subjects

Langeberg, C. J.

Publications and source records attributed to Langeberg, C. J..

9 recordsLinked to original sources

bioRxiv2026

A Noncontiguous Code for RNA-Guided DNA Recognition Preceded CRISPR

CRISPR-Cas systems use RNA-guided proteins for adaptive immunity through a mechanism whose origin is unknown. Here we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein more ancient than CRISPR-Cas and vrRNAs comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that base pair with nucleic acid targets using an uninterrupted sequence, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NNs of each repeat collectively specify a target that itself contains a gapped recognition sequence. Analysis of natural vrRNA targets suggests VIPR acts against competing phages. We demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that the roots of adaptive immunity lie in ancient warfare between viruses, and reveal a new logic for programmable genetic control.

molecular biology↗

bioRxiv2026

Stepwise DNA unwinding gates TnpB genome-editing activity

TnpB is a compact RNA-guided endonuclease and evolutionary ancestor of CRISPR-Cas12 that offers a promising platform for genome engineering. However, the genome-editing activity of TnpBs remains limited and its underlying determinants are poorly understood. Here, we used biochemical and single-molecule assays to examine the DNA-unwinding mechanism of Youngiibacter multivorans TnpB (Ymu1 TnpB). DNA unwinding proceeds through a discrete, long-lived partially unwound intermediate state before reaching a fully unwound open state. The open state forms inefficiently and collapses readily in the absence of negative supercoiling. An optimized variant, Ymu1-WFR, stabilizes formation of both the intermediate and open states, resulting in enhanced DNA cleavage in vitro and increased genome editing in plants. These findings identify the physical basis for the observed minimal activities of natural TnpBs, revealing how stabilizing specific unwinding states enables efficient DNA targeting.

biochemistry↗

bioRxiv2025

Structure and evolution-guided design of minimal RNA-guided nucleases

The design of RNA-guided nucleases with properties not limited by evolution can expand programmable genome editing capabilities. However, generating diverse multi-domain proteins with robust enzymatic properties remains challenging. Here we use an artificial intelligence-driven strategy that couples structure-guided inverse protein folding with evolution-informed residue constraints to generate active, divergent variants of TnpB, a minimal CRISPR-Cas12-like nuclease. High-throughput functional screening of AI-generated variants yielded editors that retained or exceeded wild-type activity in bacterial, plant and human cells. Cryo-EM-based structure determination of the most divergent active variant revealed new stabilizing contacts in the RNA/DNA interfaces across conformational states, demonstrating the design potential of this approach. Together these results establish a strategy for creating non-natural RNA-guided nucleases and conformationally active nucleic acid binders, enlarging the designable protein space. One-sentence abstractAn evolution- and structure-conditioned model enables design of active RNA-guided nucleases with new nucleic acid contacts resolved by cryo-EM.

synthetic biology↗

bioRxiv2025

Improving RNA Secondary Structure Prediction Through ExpandedTraining Data

In recent years, deep learning has revolutionized protein structure prediction, achieving remarkable speed and accuracy. RNA structure prediction, however, has lagged behind. Although several methods have shown some success in predicting RNA secondary and tertiary structures, none have reached the accuracy observed with contemporary protein models. The lack of success of these RNA structure prediction models has been proposed to be due to limited high-quality structural information that can be used as training data. To probe this proposed limitation, we developed a large and diverse dataset comprising paired RNA sequences and their corresponding secondary structures. We assess the utility of this enhanced dataset by retraining on a deep learning model, SincFold. We find that SincFold exhibited improved generalization to some previously unseen RNA families, enhancing its capability to predict accurate de novo RNA secondary structures. The RNASSTR dataset provides a substantial advance for RNA structure modeling, laying a strong foundation for the development of future RNA secondary structure prediction algorithms.

bioinformatics↗

bioRxiv2024

RNA elements required for the high efficiency of West Nile Virus-induced ribosomal frameshifting

West Nile Virus (WNV), a member of the Flaviviridae family, requires programmed -1 ribosomal frameshifting (PRF) for translation of the viral genome. The efficiency of WNV frameshifting is among the highest observed to date. Despite structural similarities to frameshifting sites in other viruses, it remains unclear why WNV exhibits such a high frameshifting efficiency. Here we employed dual-luciferase reporter assays in multiple human cell lines to probe the RNA requirements for highly efficient frameshifting by the WNV genome. We find that both the sequence and structure of a predicted RNA pseudoknot downstream of the slippery sequence-the codons in the genome on which frameshifting occurs-are required for efficient frameshifting. We also show that multiple proposed RNA secondary structures downstream of the slippery sequence are inconsistent with efficient frameshifting. We mapped the most favorable distance between the slippery site and the pseudoknot essential for optimal frameshifting, and found the base of the pseudoknot structure likely is unfolded prior to frameshifting. Finally, we find that many mutations in the WNV slippery sequence allow efficient frameshifting, but often result in aberrant shifting into other reading frames. Mutations in the slippery sequence also support a model in which frameshifting occurs concurrent with or after translocation of the mRNA and tRNA on the ribosome. These results provide a comprehensive analysis of the molecular determinants of WNV-programmed ribosomal frameshifting and provide a foundation for the development of new antiviral strategies targeting viral gene expression.

molecular biology↗

bioRxiv2024

Tick-borne flavivirus exoribonuclease-resistant RNAs contain a 'double loop' structure

Flaviviridae viruses are human pathogens that generate subgenomic noncoding RNAs during infection using structured exoribonuclease resistant RNAs (xrRNAs) that block progression of host cells exoribonucleases. The structures of several xrRNAs from mosquito-borne and insect-specific flaviviruses have been solved, revealing a conserved fold in which a ring-like motif encircles the end of the xrRNA. However, the xrRNAs found in tick-borne and no known vector flaviviruses have distinct characteristics and their 3-D fold was unsolved. To address this, we identified subgenomic flaviviral RNA formation in the encephalitis-causing tick-borne Powassan Virus. We characterized their secondary structure using chemical probing and solved the structure of one of its xrRNAs using cryo-EM. This structure reveals a novel double loop ring element leading to a model in which the ring is remodeled upon encountering the exoribonuclease. Using bioinformatic analyses we showed that this structure is representative of a broad class of xrRNAs and defined key structural and sequence determinants of function. These discoveries reveal a conserved strategy of structure-based exoribonuclease resistance achieved through a unique topology across a viral family of key importance to global health.

microbiology↗

bioRxiv2024

RNA language models predict mutations that improve RNA function

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data1. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function2-4. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB)5. GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which overlapping triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB- derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

synthetic biology↗

bioRxiv2023

Diversity and modularity of tyrosine-accepting tRNA-like structures

Certain positive-sense single-stranded RNA viruses contain elements at their 3 termini that structurally mimic tRNAs. These tRNA-like structures (TLSs) are classified based on which amino acid is covalently added to the 3 end by host aminoacyl-tRNA synthetase. Recently, a cryoEM reconstruction of a representative tyrosine-accepting tRNA-like structure (TLSTyr) from brome mosaic virus (BMV) revealed a unique mode of recognition of the viral anticodon-mimicking domain by tyrosyl-tRNA synthetase. Some viruses in the hordeivirus genus of Virgaviridae are also selectively aminoacylated with tyrosine, yet these TLS RNAs have a different architecture in the 5 domain that comprises the atypical anticodon loop mimic. Herein, we present bioinformatic and biochemical data supporting a distinct secondary structure for the 5' domain of the hordeivirus TLSTyr compared to those in Bromoviridae. Despite forming a different secondary structure, the 5' domain is necessary to achieve robust in vitro aminoacylation. Furthermore, a chimeric RNA containing the 5' domain from the BMV TLSTyr and the 3' domain from a hordeivirus TLSTyr are aminoacylated, illustrating modularity in these structured RNA elements. We propose that the structurally distinct 5' domain of the hordeivirus TLSTyrs performs the same role in mimicking the anticodon loop as its counterpart in the BMV TLSTyr. Finally, these structurally and phylogenetically divergent types of TLSTyr provide insight into the evolutionary connections between all classes of viral tRNA-like structures.

biochemistry↗

bioRxiv2020

An Expanded Class of Histidine-Accepting Viral tRNA-like Structures

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLSHis) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate several processes important for infection, to include replication. As only five TLSHis examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLSHis, yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLSHis RNAs. We confirmed new examples as authentic TLSHis by demonstrating their ability to be histidylated in vitro, then used mutational analyses to verify a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structures and functions in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs.

biochemistry↗