Biology subjects

Cook, A. G.

Publications and source records attributed to Cook, A. G..

16 recordsLinked to original sources

bioRxiv2026

The fungal RNA-binding protein Ssd1 represses Sun4 protein abundance through recognition of 5' UTR structural and sequence elements

Regulation of protein abundance allows fungi to adapt to changing environments, regulate their growth and morphology, and react to external stresses. Ssd1 is a fungal RNA binding protein that binds mRNAs encoding cell wall remodelling proteins and regulates cell wall biogenesis. Ssd1 binding sites (SBSs) have been described by computational and biochemical analyses, but how Ssd1 recognises these sites and how that relates to regulation of protein abundance was less clear. Here, a co-crystal structure of Saccharomyces cerevisiae Ssd1 with an SBS reveals core determinants of recognition, while fluorescent reporters of Sun4, an Ssd1-regulated cell wall protein, were used to characterise structure- guided mutations. We find that Ssd1 has an extensive RNA binding site that recognises two elements of the SBS: an upstream element that forms a structural motif, and an element containing tandem CNYU sequences that engages Ssd1 in base-specific recognition. Mutations to the Ssd1 RNA binding surface prevent Ssd1-dependent repression of fluorescent reporters and show strong phenotypes in assays of cell wall stress resistance and genetic interactions with the Cbk1 kinase. Loss of repression is also observed if both SUN4 SBSs are altered. However, the presence of one SBS in the SUN4 5' untranslated region is sufficient to confer Ssd1-dependent suppression of protein abundance. Our work confirms that RNA binding is a core function of Ssd1 and is likely to inform functional analyses in fungi beyond S. cerevisiae, where Ssd1 orthologs have been identified as virulence factors in several fungal pathogens.

molecular biology↗

bioRxiv2026

The structure of the apo-PIWI HSP90 complex

PIWI proteins are members of the Argonaute family and together with piRNAs protect metazoan germlines from transposons. PIWI proteins adopt a bi-lobed architecture with a central RNA-binding channel. HSP90 function has been linked to piRNA biogenesis, but the precise molecular mechanism is unresolved. Using the mammalian embryonic piRNA pathway as a model system, we find compelling evidence for the existence of PIWIL2- (MILI-) and PIWIL4- (MIWI2-) HSP90 complexes in foetal testis. We purify apo-PIWIL4-HSP90 from cells and determine its structure by cryo-electron microscopy. Distinct from piRNA-bound PIWI, apo-PIWIL4 adopts a unique and open conformation. The HSP90 dimer binds and unfolds PIWIs linker 1 domain. PIWIL4s N domain and the RNA-binding PAZ-MID-PIWI module are placed on opposite sides of the HSP90 dimers lumen. We further demonstrate that PIWI-HSP90 complexes, the open apo-PIWI conformation, and the HSP90 lumen-binding peptide are conserved features of PIWI proteins.

molecular biology↗

bioRxiv2026

Lysine acetylation facilitates competitive recruitment of translation termination factor eRF3a by PABPC1 C-terminal domain

In eukaryotic cells, almost all mRNA transcripts are polyadenylated in the nucleus, with the poly(A)-tail being critical for their export, translation and stability. PABPC1 is a multifunctional RNA-binding protein (RBP) that binds poly(A)-tails and is a key driver of mRNA post-transcriptional regulation. It achieves this in part by interacting with a broad range of PAM2-motif containing proteins that bind the same common site within PABPC1s PABC domain but result in diverse regulatory outcomes. Regulation of this intricate network of interactions remains poorly understood but our previous studies revealed the regulatory potential of mutually-exclusive acetylation or dimethylation of a key lysine sidechain (K606). To address this, we undertook a systematic study of PAM2-motifs, identifying translation termination factor eRF3a and translation repressor PAIP2 as having a >ten-fold higher affinity for PABPC1 relative to eleven other PAM2-motif partner proteins. Acetylation of PABPC1 at K606 specifically enhances its affinity for eRF3a in vitro, and we present the crystal structure of the specifically K606 acetylated C-terminal domain of PABPC1 in complex with the PAM2 motif of eRF3a, providing structural and functional insights into how acetylation facilitates competitive eRF3a recruitment to PABPC1 and mRNA. We further demonstrate that K606 acetylation promotes PABPC1-eRF3a interaction in cells, thereby antagonising other PAM2-dependent RNA effectors. Taken together, our results show that acetylation of lysine 606 in the PABC domain can specifically modulate PABPC1 partner selectivity, providing crucial insight into how PABPC1 multifunctionality can be regulated to coordinate mRNA usage and fate in the cytoplasm.

molecular biology↗

bioRxiv2025

DICER1 syndrome mutations lead to a gain of 3p-miRNA function, HERVH activity and increased metastatic potential

The DICER1 gene is mutated in cancer, including DICER1 syndrome, a rare tumour predisposition syndrome. Cancer-associated hotspot mutations have been reported in both catalytic domains of DICER1 and are predicted to disrupt miRNA biogenesis. To understand how these hotspot mutations contribute to cancer development, we have generated cell lines harbouring single amino acid substitutions within the catalytic RNase IIIa (S1344L) or RNase IIIb (D1709N) domains of the endogenous DICER1 gene. We show that both mutations result in a widespread loss of 5p miRNAs, and, unexpectedly, an increase in 3p passenger strands loading into AGO2. The shared similarities between both mutants can be attributed to the structural proximity of the S1344 residue to the RNase IIIb catalytic centre. Functionally, we found that changes in the repertoire of miRNAs loaded into AGO2 result in altered gene expression, impacting critical pathways for cancer development, including metastatic potential. Additionally, our results indicate that inactivating the processing activity of DICER1 does not result in genomic instability. Instead, mutations cause specific upregulation of human endogenous retrovirus H (HERVH) through miRNA-independent mechanisms, suggesting that both canonical and non-canonical DICER1 functions are important to understand DICER1 syndrome.

molecular biology↗

bioRxiv2025

A major histocompatibility complex (MHC) class II molecule that binds the same viral pathogen peptide with both nonamer and decamer core sequences for presentation to T cells

Classical molecules encoded by the major histocompatibility complex (MHC) are central to immune responses. Compared to typical mammals, the chicken MHC is small and simple, determining life or death from economically-important pathogens like avian influenza virus and Mareks disease virus (MDV). Several genes within the tightly-linked chicken MHC have been suggested to determine resistance and susceptibility to MDV, but it was a surprise to find that the dominantly-expressed class II molecule from the resistant B2 haplotype employed a novel peptide-binding mode with a decamer core sequence compared to the susceptible B19 haplotype with a typical nonamer core. We examined the crystal structure of the dominantly-expressed class II molecule from another resistant haplotype, B21, which is extremely frequent in commercial chicken flocks, to find that it bound the same MDV peptide with both nonamer and decamer cores, revealing an unexpected plasticity of binding that potentially increases the immune response to this devastating pathogen.

immunology↗

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

Integrative structural analysis of NF45-NF90 heterodimers reveals architectural rearrangements and oligomerisation on binding dsRNA.

Complexes of nuclear factors 45 and 90 (NF45-NF90) play a multitude of roles in co- and post-transcriptional RNA processing, including regulating adenosine-to-inosine editing, cassette exon and back splicing, and splicing fidelity. NF45-NF90 complexes recognise dsRNA and, in human cells, primarily interact with inverted Alu repeats (AluIRs) that are commonly inserted into introns and other non-coding RNA regions. Intronic AluIRs of [~]300 bp can regulate splicing outcomes, such as generation of circRNAs. We examined domain reorganisation of NF45-NF90 domains on dsRNAs exceeding 50 bp to gain insight into its RNA recognition properties on longer dsRNAs. Using a combination of phylogenetic analysis, solution methods (including small angle X-ray scattering and quantitative cross-linking mass spectrometry), machine learning and negative stain electron microscopy, we generated a model of NF45-NF90 complex formation on dsRNA. Our data reveal that different interactions of NF45-NF90 complexes allow these proteins to coat long stretches of dsRNA. This property of the NF45-NF90 complex has important implications for how long, nuclear dsRNAs are recognised in the nucleus and how this might promote (co)-regulation of specific RNA splicing and editing events that shape the mammalian transcriptome.

biochemistry↗

bioRxiv2024

SID-2 localises to extracellular vesicles in parasitic nematodes and does not function in environmental RNAi

In the free-living nematode Caenorhabditis elegans the transmembrane protein SID-2 imports double-stranded RNA into intestinal cells to trigger systemic RNA interference (RNAi), allowing organisms to sense and respond to environmental cues such as the presence of pathogens. This process, known as environmental RNAi, has not been observed in the most closely related parasites that are also within clade V. Previous sequence-based searches failed to identify sid-2 orthologs in available clade V parasite genomes. In this study we identified sid-2 orthologues in these parasites using genome synteny and protein structure-based comparison, following identification of a SID-2 orthologue in extracellular vesicles from the murine intestinal parasitic nematode Heligmosomoides bakeri. Expression of GFP-tagged H. bakeri SID-2 in C. elegans showed comparable localisation to the intestinal apical membrane as seen for GFP-tagged C. elegans SID-2 and further showed mobility in intestinal cells in vesicle-like structures. We tested the capacity of H. bakeri SID-2 to functionally complement environmental RNAi in a C. elegans SID-2 null mutant and show that H. bakeri SID-2 does not rescue the phenotype in this context. Therefore, our work identifies SID-2 as a highly abundant nematode EV protein whose ancestral function may be unrelated to environmental RNA and rather highlights an association with extracellular vesicle-mediated communication in free-living and parasitic nematodes.

evolutionary biology↗

bioRxiv2024

Nuclear RNA forms an interconnected network of transcription-dependent and tunable microgels

Living cells are organised through active, non-equilibrium processes, yet the physical principles governing their material states remain unclear. The cell nucleus has been described as either fluid-like or solid-like, but these behaviours have not been reconciled within a single framework. Here we show that nascent RNA and the multivalent RNA-binding protein SAF-A assemble into a nucleus-spanning microgel network maintained near the sol-gel transition. This active network exhibits scale- and time-dependent viscoelasticity and naturally reconciles solid-like and fluid-like responses. By integrating super-resolution imaging with mechanistic polymer modelling, we identify RNA production and degradation as control parameters that tune network connectivity. The model predicts enhanced susceptibility to modest perturbations near criticality, which we confirm experimentally by shifting the system between sol-like and gel-like regimes. Measurements of molecular transport, combined with simulations, reveal size-dependent diffusion and transient trapping within gel pores, establishing direct physical consequences of the near-critical state. Our results identify RNA metabolism as an energy-driven regulator of nuclear material properties and position the nucleus as a living example of an active microgel poised near criticality.

molecular biology↗

bioRxiv2023

Cell division angle regulates the tissue mechanics and tunes the amount of cerebellar folding

Modeling has proposed that the amount of neural tissue folding is set by the level of differential-expansion between tissue layers and that the wavelength is set by the thickness of the outer layer. Here we used inbred mouse strains with distinct amounts of cerebellar folding to investigate these predictions. We identified a critical period where the folding amount diverges between the strains. In this period, regional changes in the level of differential-expansion between the external granule layer (EGL) and underlying core correlate with the folding amount in each strain. Additionally, the thickness of the EGL is regionally adjusted during the critical period alongside corresponding changes in wavelength. While the number of SHH-expressing Purkinje cells predicts the folding amount, the proliferation rate in the EGL is the same between the strains. However, regional changes in the cell division angle within the EGL predicts both the tangential-expansion and thickness of the EGL. Cell division angle is likely a tunable mechanism whereby both the level of differential-expansion and thickness of the EGL are regionally tuned to set the amount and wavelength of folding.

developmental biology↗

bioRxiv2022

A network of DZF proteins controls alternative splicing regulation and fidelity

Proteins containing DZF (domain associated with zinc fingers) modules play important roles throughout gene expression, from transcription to translation. Derived from nucleotidyltransferases but lacking catalytic residues, DZF domains serve as heterodimerization surfaces between DZF protein pairs. Three DZF proteins are widely expressed in mammalian tissues, ILF2, ILF3, and ZFR, which form mutually exclusive ILF2-ILF3 and ILF2-ZFR heterodimers. Using eCLIP-Seq, we find that ZFR binds across broad intronic regions to regulate the alternative splicing of cassette and mutually exclusive exons. ZFR preferentially binds dsRNA in vitro and is enriched on introns containing conserved dsRNA elements in cells. Many splicing events are similarly altered upon depletion of any of the three DZF proteins; however, we also identify independent and opposing roles for ZFR and ILF3 in alternative splicing regulation. Along with widespread involvement in cassette exon splicing, the DZF proteins control the fidelity and regulation of over a dozen highly validated mutually exclusive splicing events. Our findings indicate that the DZF proteins form a complex regulatory network that leverages dsRNA binding by ILF3 and ZFR to modulate splicing regulation and fidelity.

molecular biology↗

bioRxiv2022

Structural analysis of SALL4 zinc-finger domain reveals a link between AT-rich DNA binding and Okihiro syndrome

Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.

biochemistry↗

bioRxiv2021

Missense variants reveal functional insights into the human ARID family of gene regulators

Missense variants are alterations to protein coding sequences that result in amino acid substitutions. They can be deleterious if the amino acid is required for maintaining structure or/and function, but are likely to be tolerated at other sites. Consequently, missense variation within a healthy population can mirror the effects of negative selection on protein structure and function, such that functional sites on proteins are often depleted of missense variants. Advances in high-throughput sequencing have dramatically increased the sample size of available human variation data, allowing for population-wide analysis of selective pressures. In this study, we developed a convenient set of tools, called 1D-to-3D, for visualizing the positions of missense variants on protein sequences and structures. We used these tools to characterize human homologues of the ARID family of gene regulators. ARID family members are implicated in multiple cancer types, developmental disorders, and immunological diseases but current understanding of their mechanistic roles is incomplete. Combined with phylogenetic and structural analyses, our approach allowed us to characterise sites important for protein-protein interactions, histone modification recognition, and DNA binding by the ARID proteins. We find that comparing missense depletion patterns among paralogs can reveal sub-functionalization at the level of domains. We propose that visualizing missense variants and their depletion on structures can serve as a valuable tool for complementing evolutionary and experimental findings.

bioinformatics↗

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

Repeated evolution of inactive pseudonucleases in a fungal branch of the Dis3/RNase II family of nucleases

The RNase II family of 3'-5' exoribonucleases are present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like "pseudonucleases". These function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. Here, we show how these pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. The N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the non-nuclease function require this region. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid "titan" growth stages and yet retains an active site sequence signature. We propose that that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, while the RNase activity was lost repeatedly in independent lineages.

molecular biology↗

bioRxiv2019

Architecture of the Mto1/2 microtubule nucleation complex

Proteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators of{gamma} -tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of the Schizosaccharomyces pombe CM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complex in vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimers in vitro and that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutants in vivo.

cell biology↗