Biology subjects

Tucker, J.

Publications and source records attributed to Tucker, J..

6 recordsLinked to original sources

bioRxiv2026

tRNA-derived fragments restrict KSHV lytic replication through miRNA-like repression of host pro-viral signalling networks

Transfer RNA derived fragments (tRFs) are emerging regulators of gene expression. Despite mounting evidence of their functional relevance in cellular and pathological processes, their roles in virus infection remain poorly understood. Here, we show that host tRFs are dynamically reprogrammed during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication and function as potent antiviral effectors. Transcriptome-wide profiling revealed extensive remodelling of the tRF landscape following viral reactivation, identifying a subset of tRFs; tRFs (tRF-His-GTG, tRF-Gly-CCC, and tRF-Lys-CTT) that are robustly induced independently of parental tRNA abundance. Functional analyses demonstrate that these tRFs suppress viral gene expression and infectious virion production. Mechanistically, we show that tRF-His-GTG and tRF-Lys-CTT engage canonical RNA silencing pathways by associating with AGO2 complexes and mediating sequence-specific repression of host target genes. Among these targets, we identify SHF as a critical pro-viral factor that promotes PI3K/Akt signalling, cellular survival and efficient viral replication. Collectively, our findings define a previously uncharacterised host defence pathway in which tRFs restrict KSHV replication by targeting host factors required for efficient viral propagation, thus expanding the functional repertoire of non-coding RNAs in antiviral immunity.

microbiology↗

bioRxiv2026

Amino acid variants at the P94 position in Staphylococcus aureus class A sortase modulate substrate binding and enzyme activity

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.

biochemistry↗

bioRxiv2025

The encoding of interoceptive-based predictions by the paraventricular nucleus of the thalamus D2+ neurons

Understanding how the brain integrates internal physiological states with external sensory cues to guide behavior is a fundamental question in neuroscience. This process relies on interoceptive predictions, which are internal models that anticipate changes in the bodys physiological state based on sensory inputs and prior experiences. Despite recent advances in identifying the neural substrates of interoceptive predictions, the precise neuronal circuits involved remain elusive. In our study, we demonstrate that Dopamine 2 Receptor (D2R+) expressing neurons in the paraventricular nucleus of the thalamus (PVT) play key roles in interoception and interoceptive predictions. Specifically, these neurons are engaged in behaviors leading to physiologically relevant outcomes, with their activity highly dependent on the interoceptive state of the mice and the expected outcome. Furthermore, we show that chronic inhibition of PVTD2R+ neurons impairs the long-term performance of interoceptive-guided motivated behavior. Collectively, our findings provide insights into the role of PVTD2R+ neurons in learning and updating state-dependent predictions by integrating past experiences with current physiological conditions to optimize goal-directed behavior.

neuroscience↗

bioRxiv2024

Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes

Facultative heterochromatinization of genomic regulators by Polycomb repressive complex (PRC) 1 and 2 is essential in development and differentiation; however, the underlying molecular mechanisms remain obscure. Using genetic engineering, molecular approaches, and live-cell single-molecule imaging, we quantify the number of proteins within condensates formed through liquid-liquid phase separation (LLPS) and find that in mouse embryonic stem cells (mESCs), approximately 3 CBX2 proteins nucleate many PRC1 and PRC2 subunits to form one non-stoichiometric condensate. We demonstrate that sparse CBX2 prevents Polycomb proteins from migrating to constitutive heterochromatin, demarcates the spatial boundaries of facultative heterochromatin, controls the deposition of H3K27me3, regulates transcription, and impacts cellular differentiation. Furthermore, we show that LLPS of CBX2 is required for the demarcation and deposition of H3K27me3 and is essential for cellular differentiation. Our findings uncover new functional roles of LLPS in the formation of facultative heterochromatin and unravel a new mechanism by which low-abundant proteins nucleate many other proteins to form compartments that enable them to execute their functions.

molecular biology↗

bioRxiv2020

Intracellular Cryptococcus neoformans disrupts the transcriptome profile of M1- and M2-polarized host macrophages

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, we used an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-{kappa}B (NF-{kappa}B) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, we identified common gene expression changes that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes.

immunology↗

bioRxiv2020

The conserved herpesviral kinase ORF36 activates B2 retrotransposons during murine gammaherpesvirus infection

Short interspersed nuclear elements (SINEs) are RNA polymerase III (RNAPIII) transcribed, retrotransposable noncoding RNA (ncRNA) elements ubiquitously spread throughout mammalian genomes. While normally silenced in healthy somatic tissue, SINEs can be induced during infection with DNA viruses, including the model murine gammaherpesvirus MHV68. Here, we explored the mechanisms underlying MHV68 activation of SINE ncRNAs. We demonstrate that lytic MHV68 infection of B cells, macrophages and fibroblasts leads to robust activation of the B2 family of SINEs in a cell autonomous manner. B2 ncRNA induction requires neither host innate immune signaling factors nor involvement of the RNAPIII master regulator Maf1. However, we identify MHV68 ORF36, the conserved herpesviral kinase, as playing a key role in B2 induction during lytic infection. SINE activation is linked to ORF36 kinase activity and can also be induced by HDAC1/2 inhibition, which is one of the known ORF36 functions. Collectively, our data suggest that ORF36-mediated changes in chromatin modification contribute to B2 activation during MHV68 infection, and that this activity is conserved in other herpesviral protein kinase homologs. AUTHOR SUMMARYViral infection dramatically changes the levels of many types of RNA in a cell. In particular, certain oncogenic viruses activate expression of repetitive genes called retrotransposons, which are normally silenced due to their ability to copy and spread throughout the genome. Here, we established that infection with the gammaherpesvirus MHV68 leads to a dramatic induction of a class of noncoding retrotransposons called B2 SINEs in multiple cell types. We then explored how MHV68 activates B2 SINEs, revealing a role for the conserved herpesviral protein kinase ORF36. Both ORF36 kinase-dependent and kinase-independent functions contribute to B2 induction, perhaps through ORF36 targeting of proteins involved in controlling the accessibility of chromatin surrounding SINE loci. Understanding features underlying induction of these elements following MHV68 infection should provide insight into core elements of SINE regulation, as well as dis-regulation of SINE elements associated with disease.

molecular biology↗