Biology subjects

Rasko, J. E.

Publications and source records attributed to Rasko, J. E..

8 recordsLinked to original sources

bioRxiv2026

Enhancing adeno-associated virus cellular entry through receptor engineering

Adeno-associated viruses (AAV) are approved for gene therapy of several genetic disorders; however, key aspects of AAV cellular entry remain poorly understood. We previously identified carboxypeptidase D (AAVR2) as an AAV receptor distinct from the multi-serotype AAV receptor KIAA0319L (AAVR). In this study, we investigate the molecular mechanisms and biological roles of AAVR and AAVR2 in mediating AAV gene transfer. Using proximity-dependent biotin identification (BioID), we defined the interactome of AAVR in the presence or absence of AAV8 and identified various proteins involved in viral entry including AAVR2. We confirmed a direct physical interaction between AAVR and AAVR2, mediated by non-AAV interacting regions in the C-termini. Further, we identified functional motifs within the carboxy-terminal tails of both receptors to facilitate the engineering of chimeric receptors with enhanced activity. Functional assays demonstrated that the overexpression of AAVR or AAVR2 enhances the cellular attachment and entry of AAV in a serotype-specific manner. Finally, we generated a stable cell line expressing a minimal functional AAVR2 with increased sensitivity for in vitro potency testing for AAVR2-engaging serotypes like AAV8. Collectively, these findings reveal significant functional similarities in AAV receptor biology and establish a framework for engineering receptor-guided modalities.

molecular biology↗

bioRxiv2025

The ZNF512B-NuRD complex co-ordinates a neural-specific gene expression program

The nucleosome remodelling and deacetylase complex (NuRD) plays a key role in chromatin regulation and a wide range of biological processes including development, haemopoiesis, immunity and neurogenesis. Its interaction with tissue-enriched and sequence-specific transcription factors (TFs) leads to distinct functional outputs in the given tissue by targeting a specific set of genes. However, how NuRD dynamically and specifically regulates gene expression in a tissue-specific manner is poorly understood. Here, we refine an N-terminal specific NuRD interaction motif which enables direct engagement with many transcriptional regulatory proteins. Using a series of structural modelling and biochemical techniques, we show that ZNF512B, a poorly characterised neuronal-expressed zinc finger protein, directly binds to the RBBP4 subunit of the NuRD complex. Subsequent knockdown of ZNF512B results in the downregulation of several neural-related molecular pathways suggesting that ZNF512B may play a regulatory role during neurogenesis. We also show that in NTERA-2 neural cells, the expression of ZNF512B is necessary for cell growth and survival, and is markedly enhanced during neural progenitor cell (NPC) differentiation. In summary, our data suggest that ZNF512B might regulate neural-specific transcriptional programs via engagement with the NuRD complex.

molecular biology↗

bioRxiv2024

Intrinsically Disordered Regions Define Unique Protein Interaction Networks in CHD Family Remodelers

Chromodomain helicase DNA-binding (CHD1-9) enzymes reposition nucleosomal DNA for transcription, recombination, and replication. They possess highly conserved ATPase domains flanked by poorly characterised N- and C-termini, which are enriched with intrinsically disordered regions (IDRs) and short aggregation-prone regions (APRs). The roles of IDRs and APRs in CHD function has remained elusive. Here, by integrating proteomics and AlphaFold Multimer analysis, we defined the protein-protein interaction (PPI) networks within the N- and C-termini of all CHDs. We generated a comprehensive map of CHD1-9-specific binding proteins, revealing dozens of novel interactions with transcription regulators. We identified APR regions that contribute to PPI formation and demonstrated that a highly conserved APR within the C-terminus of CHD4 is critical for its interaction with the nucleosome remodeling and deacetylase (NuRD), as well as the CHD, ADNP, and HP1 (ChAHP) complexes. Further analysis unravels a regulatory role for the CHD4 APR in gene transcription during erythrocyte formation. Our results emphasize that the N- and C-termini of CHD chromatin remodelers establish PPI networks that drive unique transcriptional programs.

biochemistry↗

bioRxiv2022

RNA m6A and 5hmC regulate monocyte and macrophage gene expression programs

BackgroundRNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. To date, most studies have focused on m6A, while other RNA modifications, including 5hmC, remain poorly characterised. The interplay between different RNA modifications that may occur in specific cellular contexts remains similarly unexplored. ResultsWe profiled m6A and 5hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. We observed that decreased expression of m6A and 5hmC writers, METTL3 and TET-enzymes respectively, facilitated monocyte-to-macrophage differentiation. Despite a global trend of m6A and 5hmC loss during macrophage differentiation, enrichment of m6A and/or 5hmC on specific categories of transcripts essential for macrophage differentiation positively correlated with their expression and translation. m6A and 5hmC mark and are associated with the expression of transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discovered the coexistence of m6A and 5hmC marking alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of transcripts with key roles in macrophage biology. In specific examples, RNA 5hmC controls the decay of transcripts independently of m6A. ConclusionsThis study: i) uncovers m6A, 5hmC and their writer enzymes as regulators of monocyte and macrophage gene expression programs and ii) provides a comprehensive dataset to interrogate the role of RNA modifications in a plastic system. Altogether, this work sheds light on the role of RNA modifications as central regulators of effector cells in innate immunity.

molecular biology↗

bioRxiv2022

SpliceWiz: easy, optimized, and accurate alternative splicing analysis in R

Despite an abundance of publicly available RNA sequencing datasets, a lack of integrated user-friendly tools hinder exploration of alternative splicing. SpliceWiz is an innovative, ultra-fast graphical R application that accurately quantifies splicing events using isoform-specific alignments. It is designed to accommodate hundreds of samples typically seen in clinical datasets. Novel event filters remove low-confidence measurements from analysis, enhancing accuracy over existing methods. Group-averaged strand-specific sequencing coverage plots enable clear visualization of group differences in alternative splicing, using a new file format with demonstrable performance improvements over the current BigWig standard. ompBAM, a C++ library upon which SpliceWiz is built, automates multi-threaded alignment file processing for R package developers. SpliceWiz is a powerful platform for diverse users to explore alternative splicing in large datasets.

bioinformatics↗

bioRxiv2021

Somatic mutations in CTCF zinc fingers produce cellular phenotypes explained by structure-function relationships

BackgroundHuman cancers commonly contain mutations in transcription factors that lead to aberrant DNA binding or altered effector function at target sites. One such factor significantly mutated in cancer is the evolutionarily-conserved CCCTC-binding factor (CTCF), which has fundamental roles in maintaining chromatin architecture and transcriptional regulation. Numerous cancer genome sequencing and functional studies have revealed CTCFs role as a haploinsufficient tumour suppressor gene. However, to date, structure-function relationships of somatic CTCF mutations have not been examined. MethodsWe collated somatic CTCF mutations from cancer genome portals and published studies to determine their nature, frequency, distribution and potential functional impact. We undertook an in-depth examination of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. We performed functional analyses including cell growth, colony-formation, chromatin immunoprecipitation and transcriptional reporter assays. Based on their homology, each ZF mutation was then modelled on CTCFs ZF domain crystal structure and its structural impact analysed using molecular dynamics simulations. ResultsWe observed an enrichment of somatic missense mutations occurring in the ZF region of CTCF, compared to the unstructured N- and C-termini. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. In silico mutagenesis revealed that L309P had the highest mutation energy and thus most severe impact on protein stability. Molecular docking and molecular dynamics simulations confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding (R339Q, R339W, R377H). Remarkably, R339Q and G420D were stabilised by the formation of new primary DNA bonds. All mutations exhibited some loss or gain of bonds at neighbouring residues, often in adjacent zinc fingers. ConclusionsOur data confirm the significant negative impact haploinsufficient CTCF ZF mutations have on its tumour suppressor function. A spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.

cancer biology↗

bioRxiv2020

Surveying Brain Tumor Heterogeneity by Single-Cell RNA Sequencing of Multi-sector Biopsies

Brain tumors are among the most challenging human tumors for which the mechanisms driving progression and heterogeneity remain poorly understood. We combined single-cell RNA-seq with multisector biopsies to sample and analyze single-cell expression profiles of gliomas from 13 Chinese patients. After classifying individual cells, we generated a spatial and temporal landscape of glioma that revealed the patterns of invasion between the different sub-regions of gliomas. We also used single-cell inferred CNVs and pseudotime trajectories to inform on the crucial branches that dominate tumor progression. The dynamic cell components of the multi-region biopsy analysis allowed us to spatially deconvolute with unprecedented accuracy the transcriptomic features of the core and those of the periphery of glioma at single cell level. Through this rich and geographically detailed dataset, we were also able to characterize and construct the chemokine and chemokine receptor interactions that exist among different tumor and non-tumor cells. This study provides the first spatial-level analysis of the cellular states that characterize human gliomas. It also presents an initial molecular map of the crosstalks between glioma cells and the surrounding microenvironment with single cell resolution.

genomics↗

bioRxiv2019

Ctcf Haploinsufficiency Mediates Intron Retention in A Tissue-specific Manner

CTCF is a master regulator of gene transcription and chromatin organization with occupancy at thousands of DNA target sites. CTCF is essential for embryonic development and somatic cell viability and has been characterized as a haploinsufficient tumor suppressor. Increasing evidence demonstrates CTCF as a key player in several alternative splicing (AS) regulatory mechanisms, including transcription elongation, regulation of splicing factors, and epigenetic regulation. However, the genome-wide impact of Ctcf dosage on AS has not been investigated. We examined the effect of Ctcf haploinsufficiency on gene expression and AS in multiple tissues from Ctcf hemizygous (Ctcf+/-) mice. Distinct tissue-specific differences in gene expression and AS were observed in Ctcf+/- mice compared to wildtype mice. We observed a surprisingly large number of increased intron retention (IR) events in Ctcf+/- liver and kidney, specifically in genes associated with cytoskeletal organization, splicing and metabolism. This study provides further evidence for Ctcf dose-dependent and tissue-specific regulation of gene expression and AS. Our data provide a strong foundation for elucidating the mechanistic role of CTCF in AS regulation and its biological consequences.

molecular biology↗