Biology subjects

Walters, K.

Publications and source records attributed to Walters, K..

9 recordsLinked to original sources

bioRxiv2026

Isoform-Specific Localization Diversifies Human MSI2 Function

Musashi-2 (MSI2) is an RNA-binding protein implicated in stem cell regulation and cancer, yet reports of its regulatory activity span translational activation, repression, and effects on mRNA stability, sometimes within the same cellular context. The basis for this diversity has remained unclear. Here, we identify alternative splicing as a determinant of MSI2 regulatory activity, acting through isoform-specific subcellular localization. Using a tethered reporter assay, we show that the canonical MSI2-328 isoform promotes translation without changing mRNA abundance. Truncation and mutation analysis identify a 40-amino-acid region (residues 194-234) as necessary and sufficient for cytoplasmic localization and necessary for translational activation. In contrast, the alternatively spliced MSI2-324 isoform localizes predominantly to the nucleus and fails to promote translation. We map this difference to an 18-amino-acid sequence introduced by an alternative 3 splice acceptor in exon 12; this sequence is sufficient to direct nuclear localization, and its removal restores MSI2-328-like cytoplasmic localization and translational activation. Consistent with these compartmental differences, the two isoforms associate with distinct protein networks, MSI2-328 with translation factors and ribosomal proteins, and MSI2-324 with chromatin and pre-mRNA processing factors. These isoform-specific activities are conserved across cell types, and the relative abundance of MSI2 isoforms shifts toward MSI2-324 in several cancers. Altogether, alternative splicing controls MSI2 subcellular localization, interaction networks, and regulatory output, providing a mechanistic framework for the context-dependent roles of MSI2 in gene regulation.

molecular biology↗

bioRxiv2025

Structures of dynamic interactors at native proteasomes by PhIX-MS and cryoelectron microscopy

Proteasome function depends on a network of transient interactions that remain structurally and functionally unresolved. We developed PhIX-MS (Photo-induced In situ Crosslinking-Mass Spectrometry), a structural proteomics workflow that stabilizes transient interactions in cells by UV-activated crosslinking to capture topological information. Applying PhIX-MS with cryo-electron microscopy (cryo-EM), we mapped redox sensor TXNL1 at the proteasome regulatory particle (RP), placing its PITH domain above deubiquitinase RPN11 and resolving its dynamic thioredoxin domain near RPN2/PSMD1 and RPN13/ADRM1, ideally located to reduce substrates prior to proteolysis. We also resolved chaperone PSMD5 bound to RP without the proteolytic core particle (CP) where its C-terminus inserts into the ATPase pore blocking CP binding. PhIX-MS and AlphaFold modeling tether ubiquitin ligase UBE3C/Hul5 along the RP placing its catalytic site above the RPN11 active site, enabling their coupled activities. Our integrative approach enables the localization of native, low-affinity protein interactions and is broadly applicable to dynamic macromolecular assemblies.

cell biology↗

bioRxiv2024

Structure of Blm10:13S proteasome intermediate reveals parallel assembly pathways for the proteasome core particle

Proteasomes are formed by chaperone-assisted assembly of core particles (CPs) and regulatory particles (RPs). The CP chaperone dimer Pba1/Pba2 binds early to proteasome subunits, and is thought to be replaced by Blm10 to form Blm10:CP, which promotes ATP-independent degradation of disordered proteins. Here, we present evidence of distinct parallel assembly pathways for CP by solving five cryo-EM structures including a Blm10:13S pre-assembly intermediate. Our data conflict with the current model of Blm10 and Pba1/Pba2 sequential activity in a single assembly pathway, as we find their CP binding is mutually exclusive and both are present on early and late assembly intermediates. CP affinity for Pba1/Pba2 is reduced during maturation, promoting Pba1/Pba2 release. We find Blm10 undergoes no such affinity switch, suggesting this pathway predominantly yields mature Blm10-bound CP. Altogether, our findings conflict with the current paradigm of sequential CP binding to instead indicate parallel assembly pathways by Pba1/Pba2 and Blm10.

biochemistry↗

bioRxiv2023

In situ spatial reconstruction of distinct normal and pathological cell populations within the human adrenal gland

The human adrenal gland consists of concentrically organized functionally distinct regions responsible for hormone production. Dysregulation of adrenocortical cell differentiation alters the proportion and organization of the functional zones of the adrenal cortex leading to disease. Current models of adrenocortical cell differentiation are based on mouse studies, but there are known organizational and functional differences between human and mouse adrenal glands. This study aimed to investigate the centripetal differentiation model in the human adrenal cortex and characterize aldosterone-producing micronodules (APMs) to better understand adrenal diseases such as primary aldosteronism. We applied spatially resolved in situ transcriptomics to human adrenal tissue sections from two individuals and identified distinct cell populations and their positional relationships. The results supported the centripetal differentiation model in humans, with cells progressing from the outer capsule to the zona glomerulosa, zona fasciculata, and zona reticularis. Additionally, we characterized two APMs in a 72-year-old female. Comparison with earlier APM transcriptomes indicated a subset of core genes, but also heterogeneity between APMs. The findings contribute to our understanding of normal and pathological cellular differentiation in the human adrenal cortex.

genomics↗

bioRxiv2023

A planar-polarized MYO6-DOCK7-RAC1 axis promotes tissue fluidification in mammary epithelia

Tissue fluidification and collective motility are pivotal in regulating embryonic morphogenesis, wound healing and tumor metastasis. These processes frequently require that each cell constituent of a tissue coordinates its migration activity and directed motion through the oriented extension of lamellipodia cell protrusions, promoted by RAC1 activity. While the upstream RAC1 regulators in individual migratory cells or leader cells during invasion or wound healing are well characterized, how RAC1 is controlled in follower cells remains unknown. Here, we identify a novel MYO6-DOCK7 axis that is critical for spatially restriction of RAC1 activity in a planar polarized fashion in model tissue monolayers. The MYO6-DOCK7 axis specifically controls the extension of cryptic lamellipodia required to drive tissue fluidification and cooperative mode motion in otherwise solid and static carcinoma cell collectives. HighlightsO_LICollective motion of jammed epithelia requires myosin VI activity C_LIO_LIThe MYO6-DOCK7 axis is critical to restrict the activity of RAC1 in a planar polarized fashion C_LIO_LIMYO6-DOCK7-RAC1 activation ensures long-range coordination of movements by promoting orientation and persistence of cryptic lamellipodia C_LIO_LIMyosin VI overexpression is exploited by infiltrating breast cancer cells C_LI

cell biology↗

bioRxiv2022

Discovery of E6AP AZUL binding to UBQLN1/2 in cells, phase-separated droplets, and an AlphaFold-NMR integrated structure

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 phase-separated droplets and E6AP interacts with UBQLN1/2 in cells. Steady-state and transfer NOE experiments indicate direct interaction of AZUL with the UBQLN1 UBA domain. Intermolecular contacts identified by NOESY data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We also identify a concentration-dependent oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA-adjacent, UBAA) that is -helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in droplets and cells.

biophysics↗

bioRxiv2022

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

RNA binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule inhibitor identified as a competitive inhibitor of Musashi(MSI)-RNA interactions. Here we show Ro potently inhibits adrenocortical steroidogenesis and viability independent of MSI2 in multiple cell lines. We identified Ro-interacting proteins using an unbiased proteome-wide approach and discovered it is broadly targeting RBPs. To confirm this finding, we leveraged the large-scale ENCODE data and found a subset of RBPs whose depletion phenocopies Ro inhibition. We conclude that Ro is a promiscuous inhibitor of multiple RBPs, many containing RRM1 domains. Moreover, we provide a general framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context.

molecular biology↗

bioRxiv2021

RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

Angiotensin II (AngII) binds to the type I angiotensin receptor in the adrenal cortex to initiate a cascade of events leading to the production of aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in our knowledge related to precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate siRNA screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. Together, these data support a model in which coordinated increases in transcription and regulated RNA decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic gene expression program that is temporally restricted to prevent aldosterone overproduction.

genomics↗

bioRxiv2018

Comprehensive functional profiling of long non-coding RNAs through a novel pan-cancer integration approach and modular analysis of their protein-coding gene association networks

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cellular processes in diseases such as cancer, although the functions of most remain poorly understood. To address this, here we apply a novel strategy to integrate gene expression profiles across 32 cancer types, and cluster human lncRNAs based on their pan-cancer protein-coding gene associations. By doing so, we derive 16 lncRNA modules whose unique properties allow simultaneous inference of function, disease specificity and regulation for over 800 lncRNAs. Remarkably, modules could be grouped into just four functional themes: transcription regulation, immunological, extracellular, and neurological, with module generation frequently driven by lncRNA tissue specificity. Notably, three modules associated with the extracellular matrix represented potential networks of lncRNAs regulating key events in tumour progression. These included a tumour-specific signature of 33 lncRNAs that may play a role in inducing epithelialmesenchymal transition through modulation of TGF{beta} signalling, and two stromal-specific modules comprising 26 lncRNAs linked to a tumour suppressive microenvironment, and 12 lncRNAs related to cancer-associated fibroblasts. At least one member of the 12-lncRNA signature was experimentally supported by siRNA knockdown, which resulted in attenuated differentiation of quiescent fibroblasts to a cancer-associated phenotype. Overall, the study provides a unique pan-cancer perspective on the lncRNA functional landscape, acting as a global source of novel hypotheses on lncRNA contribution to tumour progression.\n\nAuthor SummaryThe established view of protein production is that genomic DNA is transcribed into RNA, which is then translated into protein. Proteins play a critical role in shaping the function of each individual cell in the human body yet they represent less than 2% of human genomic sequence whilst up to 90% of the genome is transcribed. To explain this disparity, the existence of thousands of long non-coding RNAs (lncRNAs) has emerged that do not encode proteins but perform function as an RNA molecule. Most lncRNAs have yet to be assigned a specific biological role, so to address this we apply a novel computational approach to characterise the function of >800 lncRNAs through consistent association with protein coding genes across multiple cancer types. By doing so, we discover 16 \"modules\" of closely related lncRNAs that share broad functional themes, the most compelling of which consists of 12 lncRNAs that could regulate activation of specific cells neighbouring the tumour, leading to accelerated tumour progression and invasion. Overall, the study provides the most robust view of the lncRNA-protein coding gene landscape to date, adding to growing evidence that lncRNAs are key regulators of cancer, and have therapeutic potential comparable to proteins.

bioinformatics↗