Biology subjects

Flynn, R. A.

Publications and source records attributed to Flynn, R. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Unbiased, systematic surfaceome profiling of Acute Leukemia to identify novel immunotherapy targets

Cell surface proteomics provides a direct topological assessment of the outer membrane of cells and enables the capture of low abundance proteins that may be missed by whole cell proteomics. Here we present an unbiased atlas of the whole cell and surface proteomes of 25 commonly used leukemic cell lines, encompassing both lymphoid and myeloid lineages, and a variety of driver mutations. Paired-wise analysis highlights recurrent surface proteins that are not detected by whole cell proteomics. Coupling this dataset to RNA-sequencing, we also discovered genes where protein and RNA abundances are discordant. In KMT2A-rearranged AML, CD70 expression was increased across cell lines and validated in primary patient samples, supporting CD70 as a candidate therapeutic target in this disease. Several proteins are enriched in the surface proteomes but lack surface annotation, adding to the growing list of potential non-canonical cell surface proteins. These findings reveal a substantial pool of proteins absent from conventional surface annotations, including RNA-binding proteins, an emerging class of candidate immunotherapeutic targets.

molecular biology↗

bioRxiv2026

Intact and single-molecule analysis of heparan sulfate

Establishing tools to couple biological processes to a DNA sequence has transformed our ability to monitor life at the molecular scale due to the scalability, flexibility, and low cost of DNA sequencing. Key examples include DNA-protein (ChIP-seq1), RNA-protein (CLIP-seq2), protein-protein (proximity ligation assay3), and Cas-based recording of cellular events4. In contrast, this paradigm has not yet significantly enhanced studies of glycans, which are mostly limited to non-DNA based chemical and biochemical assays. While classical asparagine-linked and serine/threonine-linked glycans can be directly sequenced using mass spectrometry, glycosaminoglycans - notable players in the extracellular matrix - cannot be easily analyzed in their full-length form. Here we introduce HS-nano-seq, a generalized framework to selectively label, process, and detect features of heparan sulfate on a nanopore sequencing platform. Recognizing that heparan sulfate is biochemically analogous to a nucleic acid, we report purification techniques using rapid nucleic acid strategies and conjugation methods to couple DNA adapters, generating HS-DNA chimeras resolved as discrete species by capillary electrophoresis (CE). The CE assay can distinguish features of chain length and sulfation patterns. At the single-molecule level enabled by nanopore sensing, we classify a library of synthetic heparan sulfate standards and demonstrate that nanopore ionic current fingerprints encode sulfation-dependent structural features of individual HS chains. Analysis of intact, cell-derived HS could discriminate features of individual chains with different sulfation patterns, defining the heterogeneity of binding motifs across cell types and how cells organize and program the tethered extracellular matrix. More broadly, HS-nano-seq establishes a framework for achieving full-length readouts of ECM glycopolymers that are amenable to the same biological interrogation as nucleic acids.

biochemistry↗

bioRxiv2026

Aβ Aggregates Bind the U1 Spliceosomal Ribonucleoprotein in Alzheimer Disease Brain

Missplicing due to U1 small nuclear ribonucleoprotein (U1 snRNP) insolubilization and dysfunction has been identified in Alzheimer disease (AD) brain. Cytoplasmic aggregation and mislocalization of the U1 snRNP partly co-localizes with tau neurofibrillary tangles, and some evidence for tau-U1 binding has been identified. However, tau-U1 co-localization by immunohistochemistry is only partial, and insoluble U1 small nuclear RNA (snRNA) binding proteins correlate better with A{beta} than with tau in unbiased proteomics. While investigating the sedimentation characteristics of A{beta} aggregates capable of diffusing out of AD brain tissue, we unexpectedly found that some were bound to small RNA. A{beta} immunoprecipitation and deep sequencing revealed the U1 snRNA, with specific binding confirmed through reverse immunoprecipitation and oligonucleotide hybridization. Double immunoelectron microscopy revealed decoration of A{beta} fibrils, more than tau fibrils, with the U1-70k protein, a component of the U1 snRNP. Immunofluorescence of unfixed cryostat sections but not formalin fixed paraffin embedded sections of AD brain revealed labeling of a subset of amyloid plaques with anti-U1-70k antibodies, confirmed by RNA in situ hybridization of the U1 snRNA. We conclude that a subset of AD brain A{beta} aggregates are bound to the U1 snRNP at the edges of some amyloid plaques, explaining prior proteomics findings. These data provide a potential link between A{beta} aggregation and spliceosome dysfunction and unite A{beta} with other fibril-forming proteins across the neurodegenerative diseases whose aggregation is affected by RNA binding.

neuroscience↗

bioRxiv2026

Z-TAC enables custom and combinatorial degradation of cell surface proteins

Cell-surface degrader platforms typically require target-specific engineering and have therefore been applied to a relatively small set of protein targets. Here we report Z-TAC, a strategy that enables plug-and-play conversion of existing IgG antibodies into cell-surface protein degraders. Across multiple targets from distinct protein families, Z-TAC induced efficient and sustained degradation of both individual receptors and receptor combinations. For a multi-pass membrane receptor lacking selective antagonists, Z-TAC mediated complete receptor degradation and functional inhibition, demonstrating the ability of this platform to overcome the limitations of conventional pharmacological approaches. This study delineates a generalizable and scalable strategy for functional perturbation of the cell-surface proteome.

bioengineering↗

bioRxiv2025

DNASE1L3 surveils mitochondrial DNA on the surface of distinct mammalian cells

The extracellular space is a critical environment for discriminating self versus non-self nucleic acids and initiating the appropriate immune responses through signaling cascades to relay information about extracellular nucleic acids. Here, we provide evidence that oxidized mitochondrial DNA is tethered to the surface of select mammalian cells through cell surface proteins and heparan sulfate proteoglycans. We demonstrate that cell surface DNA accumulates in large clusters that partially overlap with domains enriched in RNA binding proteins. Finally, we show that human and murine B cell surfaces contain DNA that can be cleared by the secreted nuclease DNASE1L3, and that patients with a DNASE1L3 missense variant associated with increased risk for autoimmune disease harbor increased levels of surface DNA on B and T cells. Taken together, this work expands the scope of cell surface nucleic acid biology and provides a mechanistic link between cell surface molecules and DNA targeting in autoimmune disease.

cell biology↗

bioRxiv2025

Catch and release of sialoglycoRNAs enables sequencing-based profiling across cellular and extracellular material

Glycosylated RNAs (glycoRNAs) represent a recently discovered class of small RNAs, but their systematic characterization has been limited by reliance on metabolic chemical reporters and high RNA input requirements. Here we present rPAL sequencing (rPAL-seq), a sensitive and selective platform for de novo discovery of sialoglycoRNAs. rPAL-seq combines enhanced periodate oxidation of sialic acids with a capture-release workflow and optimized library construction using poly(A) extension coupled with template-switching reverse transcription. The method enabled reproducible profiling from less than 100 ng of input RNA, corresponding to less than 2% of the material required by previous approaches. When applied across 13 human cell lines, rPAL-seq identified lineage-associated glycoRNA patterns alongside a conserved core dominated by uridine-rich snRNAs and snoRNAs, with modification signatures implicating glycosylation on acp3U or related uridine-based modifications. Extending to extracellular vesicles and non-vesicular nanoparticles, rPAL-seq revealed secreted glycoRNA profiles distinct from those of the cellular fraction. rPAL-seq provides a robust, scalable strategy for glycoRNA profiling, opening new avenues for studying this emerging biopolymer.

molecular biology↗

bioRxiv2025

Mapping the nanoscale organization of the human cell surface proteome reveals new functional associations and surface antigen clusters

The cell surface is a dynamic interface that controls cell-cell communication and signal transduction relevant to organ development, homeostasis and repair, immune reactivity, and pathologies driven by aberrant cell surface phenotypes. The spatial organization of cell surface proteins is central to these processes. High-resolution fluorescence microscopy and proximity labeling have advanced studies of surface protein associations, but the spatial organization of the complete surface proteome remains uncharted. In this study, we systematically mapped the surface proteome of human T-lymphocytes and B-lymphoblasts using proximity labeling of 85 antigens, identified from over 100 antibodies tested for binding to surface-exposed proteins. These experiments were coupled with an optimized data-independent acquisition mass spectrometry workflow to generate a robust dataset. Unsupervised clustering of the resulting interactome revealed functional modules, including well-characterized complexes such as the T-cell receptor and HLA class I/II, alongside novel clusters. Notably, we identified mitochondrial proteins localized to the surface, including the transcription factor TFAM, suggesting previously unappreciated roles for mitochondrial proteins at the plasma membrane. A high-accuracy machine learning classifier predicted over 6,000 surface protein associations, highlighting functional associations such as IL10RBs role as a negative regulator of type I interferon signaling. Spatial modeling of the surface proteome provided insights into protein dispersion patterns, distinguishing widely distributed proteins, such as CD45, from localized antigens, such as CD226 pointing to active mechanisms of regulating surface organization. This work provides a comprehensive map of the human surfaceome and a resource for exploring the spatial and functional dynamics of the cell membrane proteome.

systems biology↗

bioRxiv2024

O-glycosylation contributes to mammalian glycoRNA biogenesis

There is an increasing appreciation for the role of cell surface glycans in modulating interactions with extracellular ligands and participating in intercellular communication. We recently reported the existence of sialoglycoRNAs, where mammalian small RNAs are covalently linked to N-glycans through the modified base acp3U and trafficked to the cell surface. However, little is currently known about the role for O-glycosylation, another major class of carbohydrate polymer modifications. Here, we use parallel genetic, enzymatic, and mass spectrometry approaches to demonstrate that O-linked glycan biosynthesis is responsible for the majority of sialoglycoRNA levels. By examining the O-glycans associated with RNA from cell lines and colon organoids we find known and previously unreported O-linked glycan structures. Further, we find that O-linked glycans released from small RNA from organoids derived from ulcerative colitis patients exhibit higher levels of sialylation than glycans from healthy organoids. Together, our work provides flexible tools to interrogate O-linked glycoRNAs (O-glycoRNA) and suggests that they may be modulated in human disease.

molecular biology↗

bioRxiv2024

Cell surface ribonucleoproteins cluster with heparan sulfate to regulate growth factor signaling

Receptor-ligand interactions govern a wide array of biological pathways, facilitating a cells ability to interrogate and integrate information from the extracellular space. Here, using an unbiased genome-wide knockout screen, we identify heparan sulfate proteoglycans (HSPGs) as a major component in the organizational mechanism of cell surface glycoRNA and cell surface RNA binding proteins (csRBPs). Cleavage of mature heparan sulfate chains, knockout of N- and 6-O-sulfotransferases, overexpression of endo-6-O-sulfatases, or the addition of exogenous heparan sulfate chains with high 2-O sulfation result in marked loss in glycoRNA-csRBP clustering in U2OS cells. Functionally, we provide evidence that signal transduction by HS-dependent growth factors such as VEGF-A165 is regulated by cell surface RNAs, and in vitro VEGF-A165, selectively interacts with glycoRNAs. Our findings uncover a new molecular mechanism of controlling signal transduction of specific growth factors across the plasma membrane by the regulated assembly of glycoRNAs, csRBPs, and heparan sulfate clusters.

molecular biology↗

bioRxiv2024

Development and application of GlycanDIA workflow for glycomic analysis

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from N-glycans, O-glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N-glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N-glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/584702v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18c0400org.highwire.dtl.DTLVardef@15ada57org.highwire.dtl.DTLVardef@1b4b28forg.highwire.dtl.DTLVardef@36d150_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA

We recently identified glycoRNA--a previously undescribed glycoconjugate--which consists of RNAs modified with secretory N-glycans and presented on the cell surface. While previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging periodate oxidation and aldehyde ligation (rPAL) and Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. The sensitivity and robustness of rPAL provided the first evidence of a direct glycan-RNA linkage, and its flexibility will enable further characterization of glycoRNA biology.

molecular biology↗

bioRxiv2023

RNA binding proteins and glycoRNAs form domains on the cell surface for cell penetrating peptide entry

The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA binding proteins (RBPs) are present on the surface of living cells. These cell surface RBPs (csRBPs) precisely organize into well-defined nanoclusters that are enriched for multiple RBPs, glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell surface interaction for the cell penetrating peptide TAT. Removal of RNA from the cell surface, or loss of RNA binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.

cell biology↗

bioRxiv2023

Rapid and sensitive detection of native glycoRNAs

Chemical tools enable precise characterization of many biopolymers, including glycoconjugates. Metabolic chemical reporters enabled the discovery of glycoRNAs, however they have certain limitations due the requirement of having living cells to incorporate the modified sugar. Here we develop a periodate oxidation and aldehyde ligation method to detect and characterize native sialoglycoRNAs, termed rPAL. With optimized RNA biochemistry to enhance recovery and analysis of small RNAs, we show rPAL is at least an order of magnitude more sensitive than previous methods for detecting sialoglycoRNAs. These improvements allow rPAL to detect sialoglycoRNA from human clinical samples as demonstrated by defining the abundance and patterns of sialoglycoRNAs from sorted populations of peripheral blood mononuclear cells. The sensitivity, robustness, and flexibility of rPAL will allow greater access towards characterizing glycoRNA biology.

molecular biology↗

bioRxiv2023

LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity

RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.

genomics↗

bioRxiv2020

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit. Highlights{middle dot} ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species {middle dot} Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways {middle dot} Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function {middle dot} Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection

microbiology↗

bioRxiv2020

Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 "apical-out" organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

cell biology↗

bioRxiv2019

A memory of RPS25 loss drives resistance phenotypes

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Many cell types can nonetheless withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. However, the direct and indirect consequences from sustained alterations in ribosomal protein levels are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation.

molecular biology↗

bioRxiv2019

Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells and in vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

molecular biology↗