Biology subjects

Thompson, M. K.

Publications and source records attributed to Thompson, M. K..

8 recordsLinked to original sources

bioRxiv2026

PPM1B utilizes a trinuclear metal architecture for phosphatase activity

The metal-dependent protein phosphatase (PPM/PP2C) family regulates innate immune and cell death pathways through reversible phosphorylation. Although these enzymes contain a conserved third Mg2+/Mn2+ ion (M3) that is essential for activity, its chemical role in phosphate hydrolysis has remained unclear. Here, we report studies that reveal PPM1B promotes cell death during Pseudomonas aeruginosa infection and utilizes a trinuclear metal center in which M3 directly coordinates the substrate phosphate, positioning it for in-line SN2 hydrolysis. In addition to substrate orientation, M3 positions a water molecule to protonate the departing alkoxide, stabilizing the leaving-group. Functionally, M3 substitutes for the arginine clamp in phosphoprotein phosphatases (PPP), revealing that these evolutionarily distinct phosphatase families have converged on the same chemical strategy through fundamentally different catalytic architectures. Together, these findings define a three-metal mechanism in PPM phosphatases and identify the M3 site as a rare and potentially druggable feature for immune and infectious diseases.

biochemistry↗

bioRxiv2023

Unique Binding and Stabilization Mechanisms Employed By and Engineered Into Nanobodies.

Nanobodies are single domain antibody variants that bind an antigen with the precision and affinity of a conventional antibody at only a fraction of their size. In solving the crystal structures of our nanobody-GFP complexes and compared with other available structures, we uncover mechanism that enable nanobodies to function so efficiently and effectively as single-domain antibodies. We show that unlike conventional antibodies, a nanobody repertoire maximizes sampling of their antigen surface by binding a single antigen in at least three different orientations which can be predicted by their paratope composition. We also structurally reengineering these nanobodies to improve their antigen affinity, their stability, or both - results which also revealed the strong connection between nanobody stability and affinity. We achieved this by either directly modifying the paratope, or by altering a particular region within their third framework, which is a highly conserved area that we determined plays a role in controlling nanobody stability. Our study suggests that these unique characteristics of nanobodies allow them to interact with antigens as effectively as conventional antibodies, despite their smaller size. This understanding provides methods to facilitate optimizing, humanizing and functionalizing nanobodies, thus paving the way for their utilization in diverse areas such as research, diagnostics, and therapeutic development. Significance StatementNanobodies are a unique type of antibody fragment found in select animals, containing all its antigen binding ability reduced to a single [~]15 kDa protein. There is increasing development of nanobodies for research, diagnostics, and therapeutics, yet how nanobodies function so effectively as single domain antigen binders with the precision and affinity of conventional antibodies is unclear. In this study, we present key observations to help answer this question, where one key finding is the strong relationship between nanobody stability and antigen affinity aided by the identification of a highly conserved region in nanobodies essential for maintaining nanobody stability. This region may have been retained in nanobodies in lieu of stabilizing mechanisms induced by dimerization as seen in conventional antibodies.

biochemistry↗

bioRxiv2023

Deciphering the orthorhombic crystal structure of a novel NEIL1 nanobody with pseudo-merohedral twinning

Nanobodies or VHHs (Variable Heavy domains of Heavy chain) are single domain antibodies that comprise three antigenic complementary determining regions (CDR). Nanobodies are used in numerous scientific applications including, bio-imaging, diagnosis, therapeutics, and macromolecular crystallography. We obtained crystals of a [~]14 kDa nanobody specific for the NEIL1 DNA glycosylase (hereafter called A5) in 0.5 M ammonium sulfate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, and 1.0 M lithium sulfate monohydrate from the Crystal HT Hampton Research screen that were further optimized. Here, we describe the structure determination and refinement of the A5 crystals to a resolution of 2.1 [A]. The data collected were complicated by the presence of anisotropy and twinning, and while initial space group determination pointed to a higher apparent tetragonal crystal system, the data statistics suggested twinning, placing the crystal in an orthorhombic system. Twinning was confirmed by the Padilla and Yeates test, H-test, and Britton test based on local intensity differences with a twin fraction of 0.4. Molecular replacement produced the best solution in the orthorhombic space group P21212 with four molecules in the asymmetric unit and we were able to model over 96% of the residues in the electron density with a final Rwork and Rfree of 0.1988 and 0.2289 upon refinement. SynopsisThe crystal structure of a specific nanobody against NEIL1 was determined to 2.1 [A]. The structure was ultimately solved in an orthorhombic space group after diffraction data analysis revealed mild anisotropy as well as pseudo-merohedral twinning

biochemistry↗

bioRxiv2022

Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the Drosophila larval brain

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophila larval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggests the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.

biochemistry↗

bioRxiv2022

Systematic analysis of YFP gene traps reveals common discordance between mRNA and protein across the nervous system

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation. Brief outlineA novel high resolution and sensitive approach to systematically co-visualise the distribution of mRNAs and proteins in the intact nervous system reveals that post-transcriptional regulation of gene expression is very common. The rich data landscape is provided as a browsable resource (link), using Zegami, a cloud-based data exploration platform (link). Our solution provides a paradigm for the characterisation of post-transcriptional regulation of most genes and model systems. HighlightsO_LI196/200 (98%) Drosophila genes show discordant RNA and protein expression in at least one nervous system region C_LIO_LI137/200 (68.5%) mRNAs are present in at least one synaptic compartment C_LIO_LINovel localised mRNA and protein discovered in periphery of glial processes C_LIO_LINew paradigm for analysis of post-transcriptional regulation and data exploration C_LI

cell biology↗

bioRxiv2021

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/450133v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@10f7bf1org.highwire.dtl.DTLVardef@192214dorg.highwire.dtl.DTLVardef@c84916org.highwire.dtl.DTLVardef@1366287_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain. Highlights O_LISingle molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics C_LIO_LIThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replication C_LIO_LIGenomic RNA is stable and persistent during the initial stages of infection C_LIO_LIB.1.1.7 variant replicates more slowly than the Victoria strain C_LI

microbiology↗

bioRxiv2020

Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable "open source" design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment.

genomics↗

bioRxiv2018

Precise tuning of gene expression output levels in mammalian cells

Precise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

synthetic biology↗