Biology subjects

Tollervey, D.

Publications and source records attributed to Tollervey, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Differential reconfiguration of the RNA-interactome during low glucose stress in memory and cytotoxic T cells

Cellular adaptation to nutrient fluctuation is a fundamental biological process, crucial to cell fate, function, and survival. The choice between aerobic glycolysis and oxidative phosphorylation does not merely satisfy energetic requirements but actively shapes the cellular stress responses. Here we report that during the complex metabolic programming of CD8+ T cells, glucose utilization pathways correlate with the speed and nature of the response to glucose withdrawal. By quantitating systematic RNA-protein interactions in response to glucose withdrawal, we found that effector T cells mount acute transcriptional and post-transcriptional responses to the stress, while memory T cells exhibit slower, more limited responses. The functional dichotomy observed in T cells - between highly glycolytic cytotoxic effector cells and respiratory memory cells - exemplifies how distinct glucose utilization pathways impact immunological fate and function. Understanding the intricate interplay between metabolic modality, glucose pathways, and post-transcriptional control is crucial for deciphering environment adaptations and developing interventions in contexts ranging from immunotherapy to cancer biology.

cell biology↗

bioRxiv2026

Isotope-Free Mapping of protein:RNA Interactions Using fCRAC and trxtools

Defining high-confidence RNA interaction sites for specific proteins is essential to understand RNA biology, but existing methods face trade-offs between specificity, sensitivity, and experimental accessibility. Here, we present fluorescent Cross-linking and analysis of cDNAs (fCRAC), a mammalian-cell optimized update to the CRAC protocol. In fCRAC, a fluorescent adaptor is used, in place of radiolabeling, to visualize RNA-protein complexes during gel-purification. fCRAC retains the tandem affinity purification and stringent, denaturing conditions of classical CRAC, enabling nucleotide-resolution mapping of protein:RNA interactions with high signal to noise ratio. We initially tested fCRAC using RPP25L, a component the RNase MRP and RNase P complexes. RPP25L almost exclusively bound to predicted, single sites in the RNA components (RMRP and RPPH1), showing excellent selectivity with nucleotide resolution. To support analysis of UV cross-linking data for more complex targets, we developed the trxtools package and example pipeline for standardized processing, quality control, and analysis of data from fCRAC and related methods. We include tailored strategies for repetitive RNA classes, such as tRNA and rRNA, which can be challenging to analyze using other approaches. We applied fCRAC and trxtools to define the RNA interactome of human CYCLON/CCDC86, a nuclear protein previously implicated in oncogenesis. This revealed specific interactions with rRNA, tRNA and ncRNAs involved in pre-rRNA and pre-tRNA processing. HighlightsO_LINucleotide-resolution definition of RNAs interacting with specific proteins, including rRNA and tRNA C_LIO_LIStringent denaturing purifications and robust visualization steps, with no requirement for radioactive labelling C_LIO_LITrxtools provides an integrated analysis pipeline with approaches for analyzing both single and multi-copy RNA species C_LI

molecular biology↗

bioRxiv2026

CYCLON is a nucleolar protein that regulates pan-cancer cell fitness through ribosome biogenesis

Tumor progression is driven by cancer cell fitness, defined as the capacity of malignant cells to maintain growth, adapt to stress and withstand therapy Cellular fitness is fundamentally governed by nucleolar processes, which act as central regulators by integrating RNA processing with ribosome biogenesis to support protein synthesis and stress adaptation. The nuclear protein CYCLON, containing a large intrinsically disordered region (IDR) could be implicated in mediating biomolecular condensates and regulatory plasticity, which are key elements of nucleolar biology. CYCLON also emerged as a candidate regulator of cancer cell fitness, as it is frequently overexpressed across tumor types. Inducible silencing and cell biology approaches have shown that CYCLON maintains nucleolar integrity, controls nucleoli size and number, nucleolin and Ki-67 distribution and prevents nucleolar stress. CYCLON contributes to ribosome biogenesis by binding ribosomal RNA (rRNA) and regulating 32S and 21S pre-rRNA processing, ultimately influencing ribosomal subunit production and global protein synthesis. Its depletion impairs proliferation and clonogenic capacity by prolonging both interphase and mitosis, leading to slowed cell cycle progression. The impact of CYCLON on cellular fitness has been consistently observed across cancer models, reinforcing its essential role in the regulation of nucleolar biology.

cancer biology↗

bioRxiv2025

Metabolic tuning enables immediate adaptation to energy stress in yeast

In Saccharomyces cerevisiae, glucose depletion induces metabolic reprogramming through widespread transcriptional and translational reorganization. We report that initial, very rapid translational silencing is driven by a specialized metabolic mechanism. Following glucose withdrawal, intracellular NTP levels drop drastically over 30 sec, before stabilizing at a regulated, post-stress set-point. Programmed translational control results from the differential NTP affinities of key enzymes; ATP falls below the (high) binding constants for DEAD-box helicase initiation factors, including eIF4A, driving mRNA release and blocking 80S assembly. Contrastingly, GTP levels always greatly exceed the (low) binding constants for elongation factors, allowing ribosome run-off and orderly translation shutdown. Translation initiation is immediately lost on all pre-existing mRNAs, before being preferentially re-established on newly synthesized, upregulated stress-response transcripts. We conclude that enzymatic constants are tuned for metabolic remodeling. This response counters energy depletion, rather than being glucose-specific, allowing hierarchical inhibition of energy-consuming processes on very rapid timescales.

molecular biology↗

bioRxiv2024

Pin4 Links Post-transcriptional and Transcriptional Responses to Glucose Starvation in Yeast

Adaptation to environmental change is essential in all organisms, with RNA-binding proteins (RBPs) playing critical roles in rapid cellular responses. We analyzed the largely uncharacterized yeast RBP Pin4, and its involvement in adaptation to glucose depletion. UV crosslinking (reCRAC) revealed that in glucose conditions Pin4 binds mRNA 3 UTRs with preference for a specific motif in mRNAs involved in glycolysis, amino acid, and mitochondrial metabolism. Following glucose withdrawal, Pin4-RNA binding was greatly reduced, with residual binding favoring transcripts associated with protein translation. Cells lacking Pin4 were greatly impaired in recovery from nutrient starvation and hypersensitive to oxidative stress, consistent with the mRNA targets. RNAseq and reporter assays indicated that loss of Pin4 correlated with some increases in target mRNA abundance. In wildtype yeast, glucose depletion induces diauxic shift, with massive changes in transcription patterns. Unexpectedly, this response was almost entirely abolished in cells lacking Pin4, its C-terminal prion-like domain, the RNA-recognition motif (RRM) or with an RRM point mutation. Pin4 is implicated in sensing energy depletion, which also occurs during the approach to stationary phase. We postulate that Pin4 helps coordinate post-transcriptional and transcriptional responses to energy stress, via riboregulation.

cell biology↗

bioRxiv2024

reCRAC: A Stringent Method for Precise Mapping of Protein-RNA Interactions in Yeast

Intricate interactions between RNA-binding proteins (RBPs) and RNA play pivotal roles in cellular homeostasis, impacting a spectrum of biological processes vital for survival. UV crosslinking methods to study protein-RNA interactions have been instrumental in elucidating their interactions but can be limited by degradation of target proteins during the process, low signal-to-noise ratios, and non-specific interactions. Addressing these limitations, we describe reCRAC (reverse CRAC), a novel adaptation of the CRAC (crosslinking and analysis of cDNA) technique, optimized for yeast Saccharomyces cerevisiae. Like CRAC, reCRAC applies tandem affinity purification to yield highly enriched protein preparations. However, reCRAC is redesigned to work with unstable proteins. This is achieved by lysing the cells directly into highly denaturing buffer conditions, followed by stringent purification steps. The reCRAC method was successfully applied to the easily degraded yeast protein Pin4, allowing identification of precise binding sites at base-pair resolution with greatly reduced target protein degradation and improved signal-to-noise ratios.

molecular biology↗

bioRxiv2024

The transcriptional and translational landscape of HCoV-OC43 infection

The coronavirus HCoV-OC43 circulates continuously in the human population and is a frequent cause of the common cold. Here, we generated a high-resolution atlas of the transcriptional and translational landscape of OC43 during a time course following infection of human lung fibroblasts. Using ribosome profiling, we quantified the relative expression of the canonical open reading frames (ORFs) and identified previously unannotated ORFs. These included several short upstream ORFs and a putative ORF nested inside the M gene. In parallel, we analyzed the cellular response to infection. Endoplasmic reticulum (ER) stress response genes were transcriptionally and translationally induced beginning 12 and 18 hours post infection, respectively. By contrast, conventional antiviral genes mostly remained quiescent. At the same time points, we observed accumulation and increased translation of noncoding transcripts normally targeted by nonsense mediated decay (NMD), suggesting NMD is suppressed during the course of infection. This work provides resources for deeper understanding of OC43 gene expression and the cellular responses during infection.

microbiology↗

bioRxiv2023

Transcription termination by RNA polymerase I

Transcription elongation is stochastic and driven by a Brownian ratchet mechanism, making it subject to changes in velocity. However, on regions occupied by multiple polymerases, notably the rDNA, DNA rotation plus torsion constrain polymerase molecules to proceed at the same rate generating "torsional entrainment". We report that release of entrainment, by co-transcriptional 3-end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5-exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by RNAPI subunit Rpa12. These activities were reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites, are targeted by both the exosome cofactor TRAMP and Rat1, promoting termination. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Biophysical modeling supported substantial ([~]10%) premature termination. HighlightsNascent pre-rRNA 3 cleavage promotes RNAPI deceleration and termination RNAPI undergoes early, start-site proximal termination at sites of polymerase pausing Biophysical modeling indicates [~]10% early termination - or [~]100 events per minute Model presented for overall organization of pre-rRNA transcription

molecular biology↗

bioRxiv2023

Roles of SNORD115 and SNORD116 ncRNA clusters in neuronal differentiation

Prader-Willi syndrome shows features linked to brain development and hypothalamus-related endocrine abnormalities. The smallest clinical deletions fall within the large ([~]650Kb) SNHG14 gene, removing 29 consecutive introns that each generate SNORD116. SNHG14 also includes 48 tandem introns encoding SNORD115 and generates multiple, extended snoRNA-related species. SNORD115 and SNORD116 resemble box C/D small nucleolar RNAs (snoRNAs) but lack known targets. Both snoRNAs strongly accumulated during neuronal differentiation. SNORD116 accumulation apparently reflected stabilization, potentially linked to the appearance of FBLL1, a homologue of the ubiquitous snoRNA-associated protein Fibrillarin (FBL). In contrast, SNORD115 was selectively transcribed, apparently due to regulated termination. For functional characterization we created cell lines lacking only the expressed, paternal, SNORD115 or SNORD116 cluster. Analyses during neuronal development indicated changes in RNA stability and protein synthesis. Altered mRNAs included MAGEL2, mutation of which causes the PWS-like disorder Schaaf-Yang syndrome. Comparison of SNORD115 and SNORD116 mutants indicated overlapping or interacting functions. Most changes in mRNA and protein abundance appeared relatively late in development, with roles including cytoskeleton formation, extracellular matrix, neuronal arborization. Comparison with human embryonic midbrain development suggested enhanced progression in neuronal development in the snoRNA mutants. Subtle impairment of relative neuronal maturation during development, might generate the clinical phenotypes.

cell biology↗

bioRxiv2023

Improved detection and quantitation of RNA-interactomes using DIA SILAC

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time.

molecular biology↗

bioRxiv2022

Posttranscriptional regulation of cell wall integrity in budding yeast

The fungal cell wall provides protection and structure, and is an important target for antifungal compounds. A MAP kinase cascade termed the cell wall integrity (CWI) pathway regulates transcriptional responses to cell wall damage. Here we describe a posttranscriptional pathway that plays an important complementary role. We discovered that the RNA-binding proteins (RBPs) Mrn1 and Nab6 specifically target the 3 UTRs of a largely overlapping set of cell wall-related mRNAs. These mRNAs are downregulated in the absence of Nab6, indicating a function in target mRNA stabilization. Nab6 acts in parallel to CWI signaling to maintain appropriate expression of cell wall genes during stress. Cells lacking both pathways are hypersensitive to antifungal compounds targeting the cell wall. Deletion of MRN1 partially alleviates growth defects associated with{Delta} nab6 and Mrn1 has an opposing function in mRNA destabilization. Our results uncover a novel posttranscriptional pathway which mediates cellular resistance to antifungal compounds.

cell biology↗

bioRxiv2022

The SPARC complex defines RNAPII promoters in Trypanosoma brucei

Kinetoplastids are a highly divergent lineage of eukaryotes with unusual mechanisms for regulating gene expression. We previously surveyed 65 putative chromatin factors in the kinetoplastid Trypanosoma brucei. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). Here we report that SET27 and CRD1, together with four previously uncharacterized constituents, form the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Transcriptome analysis in the absence of SET27 revealed upregulated mRNA expression in the vicinity of SPARC peaks within the main body of chromosomes in addition to derepression of genes encoding variant surface glycoproteins (VSGs) located in subtelomeric regions. These analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality.

molecular biology↗

bioRxiv2022

EZH2 interacts with MEG3 to directly regulate integrin signaling and endothelial cell function

Enhancer of Zeste Homologue 2 (EZH2) modulates gene transcription during endothelial cell (EC) dysfunction, via interaction with non-coding RNAs (ncRNAs). Thus, EZH2 can act as a rheostat in deposition of histone H3K27 trimethylation (H3K27me3) to repress many genes. We profiled EZH2-RNA interactions using formaldehyde/UV assisted cross-linking ligation and sequencing of hybrids (FLASH-seq) in primary human ECs. Transcriptome-wide EZH2-associated ncRNAs and RNA-RNA interactome were obtained. This approach revealed EZH2 directly binding maternally expressed gene (MEG3) and MEG3:MEG3 hybrid structures. By chromatin immunoprecipitation with sequencing (ChIP-seq) following depletion of MEG3, we discovered that MEG3 targets and controls recruitment of EZH2/H3K27me3 onto a regulatory region of integrin subunit alpha 4 (ITGA4). MEG3 knockdown or pharmacological inhibition of EZH2 de-repressed ITGA4, whilst improving endothelial cell function in vitro, and increasing ITGA4 expression in vivo. Our study demonstrates new role for MEG3, as instrumental in epigenetic regulation of EC function by EZH2, through targeting of integrin-dependent signalling.

molecular biology↗

bioRxiv2022

The RNA-binding landscape of HAX1 protein indicates its involvement in ribosome biogenesis and translation.

HAX1 is a human protein with no known homologues or structural domains, mutations in which cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here we report transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in ribosome biogenesis and rRNA processing. Using CRISPR knockouts we find that RNA targets of HAX1 partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. Functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

molecular biology↗

bioRxiv2021

Systematic mapping of small nucleolar RNA targets in human cells

Altered expression of box C/D small nucleolar RNAs (snoRNAs) is implicated in human diseases, including cancer. Box C/D snoRNAs canonically direct site-specific, 2-O-methylation but the extent to which they participate in other functions remains unclear. To identify RNA targets of box C/D snoRNAs in human cells, we applied two techniques based on UV crosslinking, proximity ligation and sequencing of RNA hybrids (CLASH and FLASH). These identified hundreds of novel snoRNA interactions with rRNA, snoRNAs and mRNAs. We developed an informatic pipeline to rigorously call interactions predicted to direct methylation. Multiple snoRNA-rRNA interactions identified were not predicted to direct RNA methylation. These potentially modulate methylation efficiency and/or contribute to folding dynamics. snoRNA-mRNA hybrids included 1,300 interactions between 117 snoRNA families and 940 mRNAs. Human U3 is substantially more abundant than other snoRNAs and represented about 50% of snoRNA-mRNA hybrids. The distribution of U3 interactions across mRNAs also differed from other snoRNAs. Following U3 depletion, mRNAs showing altered abundance were strongly enriched for U3 CLASH targets. Most human snoRNAs are excised from pre-mRNA introns. Enrichment for snoRNA association with branch point regions of introns that contain snoRNA genes was common, suggesting widespread regulation of snoRNA maturation.

cell biology↗

bioRxiv2021

A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first processing-specific human disorder to be described. HighlightsO_LIMutations in RMRP lncRNA impair pre-rRNA processing and T cell activation C_LIO_LIPatient derived fibroblasts show impaired pre-rRNA processing C_LIO_LICells with the most common disease-linked mutation have specific processing defects C_LIO_LICytoplasmic ribosomes and intact RNase MRP complexes are also reduced in these cells C_LI

cell biology↗

bioRxiv2020

Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

The conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not well understood. Here we present the crystal structure of Ssd1 at 1.9 [A] resolution. Active RNase II enzymes have a characteristic, internal RNA binding path, but in Ssd1 this is blocked by remnants of regulatory sequences. Instead, RNA binding activity has likely been relocated to the outer surface of the protein. Using in vivo crosslinking and cDNA analysis (CRAC), we identify Ssd1-RNA binding sites. These are strongly enriched in 5UTRs of a subset of mRNAs encoding cell wall proteins. Based on these and previous analyses, we identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory elements in the RNase II family.

biochemistry↗

bioRxiv2020

Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His8, the C-terminus with His8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

cell biology↗