Biology subjects

Cate, J. H. D.

Publications and source records attributed to Cate, J. H. D..

At least 19 recordsLinked to original sources

bioRxiv2026

tRNA-derived second messengers confer bacterial immunity

Bacterial small-molecule messengers that function in diverse immune signaling pathways have been thought to derive strictly from biosynthesis using nucleotide and cofactor substrates. Here we report a nuclease-driven immune signaling pathway in which tRNA cleavage generates a specific small molecule that triggers cell death in response to RNA-guided transcript recognition in prokaryotes. We identify a class of type VI-B CRISPR systems in which the RNA-guided nuclease Cas13 and its accessory transmembrane protein, 2TM{beta}, are co-encoded in an operon and are jointly required for bacteriophage defense. Upon target RNA recognition, Cas13 activates 2TM{beta} by producing mono- and dinucleotide messengers bearing 2',3'-cyclic uridine phosphate ends by tRNA cleavage. Activated 2TM{beta} destroys infected cells by forming a multimeric complex that disrupts cell membranes to mediate cell death. These findings demonstrate RNA-programmed tRNA cleavage for second-messenger biogenesis, revealing a new logic for prokaryotic immune signaling.

microbiology↗

bioRxiv2026

Enzymatic formation of a conserved isoaspartate in ribosomal protein uS11

Isoaspartate (isoAsp) formation is typically viewed as a "molecular clock" through nonenzymatic degradation of aspartate or asparagine during protein aging. Here we report a nearly universal enzymatic pathway for the formation of a conserved isoAsp in the bacterial ribosomal protein uS11. Proteome-wide protein-protein interaction scans using AlphaFold3 identified YbeY as a candidate enzyme from Escherichia coli. NMR spectroscopy supported a stable YbeY-uS11 complex from Thermotoga maritima. Biochemical assays indicated that EcYbeY catalysis is zinc-dependent and prefers the conserved Asn-Gly motif for isoAsp formation. A high-resolution cryo-electron microscopy structure of the 70S ribosome from E. coli {Delta}ybeY revealed that loss of isoAsp alters contacts with the 16S rRNA groove and bS21. Phylogenetic analysis indicated that YbeY is present in almost all bacteria, and its absence is correlated to changes in the Asn-Gly motif of uS11. Additionally, our structural analyses implicate Fap7 as the functional counterpart in archaea and eukaryotes.

biochemistry↗

bioRxiv2026

Altering the ribosome exit tunnel to improve consecutive incorporation of challenging monomers

Ribosomes are capable of incorporating a wide array of natural and unnatural monomers into growing polymer chains, but can be stalled by monomers with constrained or non-natural backbones. Here we evaluate whether monomer-dependent ribosome stalling can be alleviated by structure-guided mutations to 23S rRNA within the exit tunnel. Ribosomes harboring an A2062U mutation are as active as wild type (WT) ribosomes when translating non-proline sequences and up to 10-fold more active when translating sequences containing up to four consecutive proline residues. High-resolution cryo-EM structures of WT and A2062U mutant ribosomes containing a polyproline nascent chain reveal that the A2062U mutation relieves an exit tunnel constriction to better accommodate a conformationally restricted peptide chain. A2062U mutant ribosomes also improve translation of sequences containing multiple, consecutive {beta}2-hydroxy acids. These results provide a mechanistic basis for engineering the ribosome for improved translation of genetically encoded polymers with novel backbones.

biochemistry↗

bioRxiv2026

A family of archaeal hibernation factors that bind in tandem and protect ribosomes in dormant cells

Under nutrient limitation or stress, ribosome hibernation factors inactivate and protect ribosomes. Although ribosome hibernation plays an important role in microbes, we lack a complete understanding of this process in archaea. Here, we identify a family of hibernation factors, which we designate as single ribosomal subunit inhibitors (SriA-SriD), from the methanogenic archaeon Methanosarcina acetivorans. All four sri genes are encoded in an operon and each Sri protein inhibits protein synthesis in vitro. Deletion of sri genes in M. acetivorans impaired growth recovery after prolonged stationary phase and also led to depletion of the small ribosomal subunit. Cryo-EM structures show that Sri proteins bind to the ribosome in tandem and form conserved protein-protein interfaces. Sri is broadly distributed across archaeal phyla and sri genes frequently co-occur. Together, these findings establish Sri proteins as a distinct group of hibernation factors that protect ribosomes during dormancy and expand our understanding of ribosome hibernation in archaea.

microbiology↗

bioRxiv2025

Translational Downregulation of 5' TOP mRNAs During T Cell Exhaustion

T cell exhaustion is a dysfunctional state that arises during chronic infections and cancer, characterized by impaired effector functions and sustained expression of inhibitory receptors. While transcriptional, epigenetic, and metabolic rewiring have been well documented in exhausted T cells, a comprehensive understanding of how translation is regulated in this state remains incomplete. To address this gap, we performed ribosome profiling and RNA sequencing on exhausted human CD8+ T cells to globally assess translational control. Our analyses reveal a marked repression of 5 terminal oligopyrimidine (TOP) mRNAs during exhaustion. Unexpectedly, we demonstrate that this translational repression occurs despite evidence of elevated mTOR activity in exhausted T cells. These findings uncover a previously unknown layer of translational control in exhausted T cells.

molecular biology↗

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

Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

Cell-free translation from human cells is a powerful platform for studying mammalian gene expression and building synthetic biology tools, but productivity is often curtailed by inhibitory phosphorylation of eIF2 on residue Ser52. Here we systematically explored complementary strategies to bypass this initiation block across editable and hard-to-edit human cell types. In Expi293F suspension cells, precise genome editing of EIF2S1 to block Ser52 phosphorylation (eIF2-S52A) produced high-activity extracts. Genetic knockout of EIF2AK2 (PKR)-the principal eIF2 kinase engaged in eIF2 phosphorylation in Expi293F lysates-also improved translation, further establishing eIF2 phosphorylation as the dominant bottleneck in Expi293F translation extracts. Because genome editing is impractical in many contexts including primary human cells, we also implemented expression-based rescue of eIF2 phosphorylation: stable piggyBac integration of truncated GADD34 (PPP1R15A) and K3L, a viral eIF2 decoy, under control of a Tet-inducible promotor in induced pluripotent stem cells (iPSCs) and primary human fibroblasts. After differentiating engineered KOLF2.1J iPSCs into cardiomyocytes, we found that stable GADD34/K3L expression increased translation output in cardiomyocyte translation extracts. Using the piggyBac expression system in primary fibroblasts also resulted in improved translational output. Together these data pinpoint eIF2 phosphorylation as the key barrier to robust translation in human cell translation extracts. They also show that editing eIF2 or removing PKR is optimal where genome editing is feasible, while providing a portable GADD34/K3L expression cassette enables production of translationally active lysates from systems where genome editing is challenging or not possible.

biochemistry↗

bioRxiv2025

miRNA-Mediated Regulation of Gene Expression During Early Activation in Jurkat Cells

BackgroundT cell activation induces substantial changes in gene expression by rapidly increasing transcription and translation. Additionally, microRNAs play a crucial role in regulating protein expression in T cell physiology, adding a layer of complexity by fine-tuning protein levels. While various miRNAs have been implicated in T cell function, a systematic analysis of differentially expressed miRNAs during early T cell activation and identification of their mRNA targets remains mostly unknown. ResultsWe investigated dynamic changes in global gene expression during early T cell activation using a multi-omics approach combining small RNA-seq, mRNA-seq and ribosome profiling. Our results show that most differential expression changes occur by 5 hours post-activation, with translational upregulation predominating over downregulation. From 5 to 12 hours, we observed modest transcriptional and translational reprogramming. We identified 9 miRNAs that are differentially expressed (DE) during early activation, with most changes occurring as early as 5 hours. We calculated translation efficiency (TE) and classified genes based on changes in both mRNA abundance and ribosome-protected fragments (RPFs). By integrating TE and miRNA expression data, we examined the relationship between TE group-specific regulation patterns and the number of miRNA binding sites. Interestingly, rather than observing a uniform downregulation of targets with 4 or more predicted DE miRNA binding sites, we found distinct regulatory patterns that varied with both activation time point and TE category. ConclusionsOur data provide new insights into how genes associated with key events in T cell activation such as translation, cell proliferation, and immune signaling are regulated at both the transcriptional and translational levels. The observation that most regulatory changes occur within the first 5 hours post-activation highlights the rapid and coordinated nature of T cell responses. The differential patterns of target regulation, based on translation efficiency groups and miRNA binding site density, suggest a context-dependent role for miRNAs in shaping protein output. Future experiments will be required to functionally validate specific miRNA-target interactions and to explore their relevance in primary T cells in vivo. This study also lays the groundwork for identifying miRNA-based regulatory circuits for therapeutic modulation of T cell activity.

genomics↗

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↗

bioRxiv2025

Circular 23S rRNA within archaeal ribosomes

The ribosomal RNAs (rRNAs) that form the core of ribosomes are believed to occur as linear molecules. Here, we investigated rRNAs from diverse and mostly uncultivated archaea and found evidence that, in at least eight phylum-level archaeal lineages, the 23S rRNAs within mature ribosomes are circular. Sequencing of total cellular RNA indicates that the transcriptional abundances of archaeal circular 23S rRNAs vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggested that the circular molecules are within the ribosomes. Thus, we directly sequenced RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirmed that the assembled 23S rRNAs are circular. Structural modeling places the 5 and 3 ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their further circularization within ribosomes. We confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups. Overall, the results uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

microbiology↗

bioRxiv2024

Structure of an Archaeal Ribosome with a Divergent Active Site

The ribosome is the universal translator of the genetic code and is shared across all life. Despite divergence in ribosome structure over the course of evolution, the peptidyl transferase center (PTC), the catalytic site of the ribosome, has been thought to be nearly universally conserved. Here, we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the Pyrobaculum calidifontis ribosome. We find that sequence variation leads to the rearrangement of key PTC base triples and differences between archaeal and bacterial ribosomal proteins also enable sequence variation in archaeal PTCs. Finally, we identify a novel archaeal ribosome hibernation factor that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome.

evolutionary biology↗

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

Role of ribosomal protein bS1 in orthogonal mRNA start codon selection

In many bacteria, the location of the mRNA start codon is determined by a short ribosome binding site sequence that base pairs with the 3'-end of 16S ribosomal RNA (rRNA) in the 30S subunit. Many groups have changed these short sequences, termed the Shine-Dalgarno (SD) sequence in the mRNA and the anti-Shine-Dalgarno (ASD) sequence in 16S rRNA, to create "orthogonal" ribosomes to enable the synthesis of orthogonal polymers in the presence of the endogenous translation machinery. However, orthogonal ribosomes are prone to SD-independent translation. Ribosomal protein bS1, which binds to the 30S ribosomal subunit, is thought to promote translation initiation by shuttling mRNA to the ribosome. Thus, a better understanding of how the SD and bS1 contribute to start codon selection could help efforts to improve the orthogonality of ribosomes. Here we engineered the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit, to separate the activity of bS1 binding to the ribosome from the role of the mRNA SD sequence in start codon selection. We find that ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore, orthogonal 30S subunits lacking bS1 do not have improved orthogonality. Our findings suggest that mRNA features outside the SD sequence and independent of bS1 binding to the ribosome likely contribute to start codon selection and the lack of orthogonality of present orthogonal ribosomes.

biochemistry↗

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

JUN mRNA Translation Regulation is Mediated by Multiple 5' UTR and Start Codon Features

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5' -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

molecular biology↗

bioRxiv2023

eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3 untranslated region (3-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

molecular biology↗

bioRxiv2023

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells,1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

cell biology↗

bioRxiv2023

Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

The Escherichia coli ribosome can incorporate a variety of non-O_SCPLOWLC_SCPLOW--amino acid monomers into polypeptide chains, but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives-3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)-the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high resolution cryo-EM structures of the ribosome with these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). These structures reveal how the aromatic ring of each monomer sterically blocks positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the "proton wire" responsible for facilitating formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a clear rationale for differences in reactivity of aminobenzoic acid derivatives relative to O_SCPLOWLC_SCPLOW--amino acids and each other, and point to stereochemical constraints on the size and geometry of non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes.

biophysics↗