Biology subjects

Yee, B. A.

Publications and source records attributed to Yee, B. A..

16 recordsLinked to original sources

bioRxiv2025

Dosage-sensitive RBFOX2 autoregulation promotes cardiomyocyte differentiation through transcriptome maturation

Haploinsufficiency of the RNA splicing regulator, RBFOX2, is linked to congenital heart disease (CHD), yet its pathogenic mechanisms remain unclear. Here, we demonstrate that RBFOX2 is essential for progressing cardiomyocyte (CM) differentiation by shifting exon usage profiles to more mature patterns in sarcomere, cytoskeletal, and focal adhesion genes, including alpha-actinin-2 (ACTN2). This maturation program is initiated by critical levels of RBFOX2 that facilitate autoregulatory splicing at mutually exclusive exons encoding early and late isoforms with distinct functional roles. In heterozygous CMs, autoregulation is disrupted, which skews isoform ratios and generates a dominant-negative product caused by exon co-inclusion. Finally, we demonstrate that overexpression of ACTN2 rescues heterozygous, but not null, phenotypes by restoring contractility, which triggers a mechanosensing feedback loop involving upregulation of RBFOX2 from the wildtype allele and transcriptome maturation. Our data suggest that decreased RBFOX2 dosage and autoregulation impair CM differentiation, contributing to CHD pathogenesis and heart failure susceptibility.

developmental biology↗

bioRxiv2025

Genome-Wide Interrogation of SARS-CoV-2 RNA-Protein Interactions Uncovers Hidden Regulatory Sites

The global impact of the COVID-19 pandemic underscores the critical need for a comprehensive understanding of SARS-CoV-2 replication mechanisms. While the central roles of the RNA dependent RNA polymerase (NSP12), primase protein (NSP8), and nucleocapsid protein (N) in the virus life cycle are extensively studied, the precise nature of their interactions with the full-length viral RNA genome remain incompletely characterized. In this study, we sought to address this knowledge gap by employing enhanced crosslinking and immunoprecipitation (eCLIP) to map the binding sites of NSP8, NSP12, and N proteins across the SARS-CoV-2 genome at early stages of viral RNA and protein synthesis and late stages of virion assembly. Our findings revealed interactions of NSP8 and NSP12 to the 5 and 3 untranslated regions (UTRs) of both positive and negative sense RNA, regions known to regulate viral replication, transcription, and translation. We identified a surprising and essential NSP12 binding site within the RNA sequence encoding the conserved Y1 domain of NSP3, which regulates RNA abundance upstream of the site. Additionally, we found that N protein interacts with the 5 UTR and influences translation efficiency. Finally, we report a novel regulatory function of N protein in modulating ribosomal frameshifting proximal to the frameshift element, a crucial process for maintaining viral protein stoichiometry. Our results provide a detailed molecular map of SARS-CoV-2 protein-RNA interactions, revealing potential therapeutic targets for attenuating viral fitness and informing the development of next-generation antiviral strategies.

molecular biology↗

bioRxiv2025

Short 5' UTRs serve as a marker for viral mRNA translation inhibition by the IFIT2-IFIT3 antiviral complex

Recognition of "non-self" nucleic acids, including cytoplasmic dsDNA, dsRNA, or mRNAs lacking proper 5 cap structures, is critical for the innate immune response to viruses. Here, we demonstrate that short 5 untranslated regions (UTRs), a characteristic of many viral mRNAs, can also serve as a molecular pattern for innate immune recognition via the interferon-induced proteins IFIT2 and IFIT3. The IFIT2-IFIT3 heterodimer, formed through an intricate domain swap structure resolved by cryo-EM, mediates viral mRNA 5 end recognition, translation inhibition, and ultimately antiviral activity. Critically, 5 UTR lengths <50 nucleotides are necessary and sufficient to sensitize an mRNA to translation inhibition by the IFIT2-IFIT3 complex. Accordingly, diverse viruses whose mRNAs contain short 5 UTRs, such as vesicular stomatitis virus and parainfluenza virus 3, are sensitive to IFIT2-IFIT3-mediated antiviral activity. Our work thus reveals a pattern of antiviral nucleic acid immune recognition that takes advantage of the inherent constraints on viral genome size.

microbiology↗

bioRxiv2025

LARP6 regulates the mRNA translation of fibrogenic genes in liver fibrosis

Metabolic syndrome and excessive alcohol consumption result in liver injury and fibrosis, which is characterized by increased collagen production by activated Hepatic Stellate Cells (HSCs). LARP6, an RNA-binding protein, was shown to facilitate collagen production. However, LARP6 expression and functionality as a regulator of fibrosis development in a disease relevant model remains elusive. By using snRNA-sequencing, we show that LARP6 is upregulated mainly in HSCs of liver fibrosis patients. Moreover, LARP6 knockdown in human HSCs suppresses fibrogenic gene expression. By integrating eCLIP analysis and ribosome profiling in HSCs, we show that LARP6 interacts with mature mRNAs comprising over 300 genes, including RNA structural elements within COL1A1, COL1A2, and COL3A1 to regulate mRNA expression and translation. Furthermore, LARP6 knockdown in HSC attenuates fibrosis development in human liver spheroids. Altogether, our results suggest that targeting LARP6 in human HSCs may provide new strategies for anti-fibrotic therapy. HighlightsO_LILARP6 is upregulated in liver fibrosis, mainly in HSCs. C_LIO_LILARP6 knockdown in human HSCs reduces liver fibrosis development. C_LIO_LIOf the hundreds of gene targets, LARP6 interacts most with collagen mRNAs. C_LIO_LILARP6 regulates mRNA translation via interaction with 5UTRs. C_LI

cell biology↗

bioRxiv2025

Stress Granules Underlie Acute Myeloid Leukemia Stem Cell Survival and Stress Adaptation

The link between cancer maintenance and an ability to sustain continued growth through stresses conferred by the cancer state itself is growing. However, there are significant gaps in our understanding of how this stress is managed, particularly at the level of cancer initiating cells. Here, we identify proteins comprising the dynamic, stress-adaptive ribonucleoprotein complexes known as stress granules (SG) to be enriched among the factors essential for leukemic stem cell (LSC)-driven leukemic propagation. Focusing on core SG nucleator G3BP1, we dissect the role of SGs in human acute myeloid leukemia (AML), their targetability, and the mechanisms they govern to uncover a novel propensity for AML, and in particular LSC-enriched fractions, to prime the expression of SG components, form SGs with greater fidelity and to be reliant on their establishment and continued integrity for LSC maintenance. We further unveil the transcript and protein interactome of G3BP1 in the AML context and show that consolidated control of innate immune signaling, and apoptosis repression is executed through regional binding specificity of G3BP1 to highly structured 3UTRs and cooperation with the RNA helicase UPF1 to mediate transcript decay in SGs. Altogether our findings advance novel fundamental principles of stress adaptation exploited in AML and LSCs that may extend to other cancers and uncover SGs as a novel axis for therapy development.

cancer biology↗

bioRxiv2023

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

microbiology↗

bioRxiv2022

Transcriptome Regulation by PARP13 in Basal and Antiviral States in Human Cells

The RNA-binding protein PARP13 is a primary factor in the innate antiviral response. PARP13 suppresses translation and drives decay of bound viral and host RNA. PARP13 interacts with many proteins encoded by interferon-stimulated genes (ISG) to activate antiviral pathways including post-translational addition of ISG15, or ISGylation. We performed enhanced crosslinking immunoprecipitation (eCLIP) and RNA-seq in human cells to investigate PARP13s role in transcriptome regulation for both basal and antiviral states. We find that the antiviral response shifts PARP13 target localization but not its binding preferences and that PARP13 supports the expression of ISGylation-related genes, including PARP13s cofactor, TRIM25. We elucidate a transcriptome-wide periodicity of PARP13 binding around TRIM25 and show they associate in part via RNA-protein interactions. Taken together, our study implicates PARP13 in creating and maintaining a cellular environment poised for an antiviral response through limiting PARP13 translation, regulating access to distinct mRNA pools, and elevating ISGylation machinery expression.

molecular biology↗

bioRxiv2022

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1/{beta}. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

molecular biology↗

bioRxiv2022

Scalable and deep profiling of mRNA targets for individual microRNAs with chimeric eCLIP

Our expanding knowledge of the roles small regulatory RNAs play across numerous areas of biology, coupled with the promise of RNA-targeted therapies and small RNA-based medicines, create an urgent need for tools that can accurately identify and quantify small RNA:target interactions at scale. MicroRNAs (miRNA) are a major class of small RNAs in plants and animals. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments in chimeric CrossLinking and ImmunoPrecipitation (CLIP) provides a direct readout of miRNA targets with high-throughput sequencing. Despite the power of this approach, widespread adoption of chimeric CLIP has been slow due to both methodological technical complexity as well as limited recovery of chimeric molecules (particularly beyond the most abundant miRNAs). Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery. We show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal- to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further demonstrate that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeting by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP-identified targets and indicated that probe-enriched chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and for revealing a deeper picture of regulatory networks for specific miRNAs of biological interest. HighlightsO_LINo-gel chimeric eCLIP improves recovery of miRNA:mRNA interactions by 70-fold C_LIO_LIProbe- and PCR-enrichment deeply profiles mRNA targets of miRNAs of interest C_LIO_LIChimeric eCLIP targets experimentally identify non-computationally predicted interactions C_LIO_LIIncreased depth recovers [~]6 million miRNA:target chimeras in HEK293T C_LI

genomics↗

bioRxiv2021

Identification of the Global miR-130a Targetome Reveals a Novel Role for TBL1XR1 in Hematopoietic Stem Cell Self-Renewal and t(8;21) AML

Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSC) point to shared core stemness properties. However, discordance between mRNA and protein signatures underscores an important role for post-transcriptional regulation by miRNAs in governing this critical nexus. Here, we identified miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impaired B lymphoid differentiation and expanded long-term HSC. Integration of protein mass spectrometry and chimeric AGO2 eCLIP-seq identified TBL1XR1 as a primary miR-130a target, whose loss of function phenocopied miR-130a overexpression. Moreover, we found that miR-130a is highly expressed in t(8;21) AML where it is critical for maintaining the oncogenic molecular program mediated by AML1-ETO. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of novel genes and molecular networks underpinning stemness properties of normal and leukemic cells. HIGHLIGHTSO_LImiR-130a is a regulator of HSC self-renewal and lineage commitment C_LIO_LITBL1XR1 is a principal target of miR-130a C_LIO_LITBL1XR1 loss of function in HSPC phenocopies enforced expression of miR-130a C_LIO_LIElevated miR-130a levels maintain the AML1-ETO repressive program in t(8;21) AML C_LI

cell biology↗

bioRxiv2021

Splicing Factor SRSF1 Deficiency in the Liver Triggers NASH-like Pathology via R-Loop Induced DNA Damage and Cell Death

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA-DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver failure.

cell biology↗

bioRxiv2020

RNA adenosine deaminase ADAR2 modulates T helper 17 cell effector function

ADAR1 and ADAR2 catalyze adenosine-to-inosine (A-to-I) editing, the most common post-transcriptional modification in RNA. While ADAR1 is ubiquitously expressed and plays a critical role in preventing activation of the host immune system, ADAR2 exhibits tissue-specific and inducible expression patterns, and its function in the immune system is not known. Here, we identify an intragenic super-enhancer involved in the dramatic induction of ADAR2 when naive helper T cells differentiate toward the Th17 lineage. By editing the inverted repeat sequences at the 3 untranslated region (UTR) of Malt1, which encodes a component of the NF-{kappa}B activation complex, ADAR2 promotes MALT1 expression and Th17 effector function. Interference with the ADAR2-MALT1 pathway dampens the production of Th17 cytokines and promotes T cell-mediated colitis. This study expands our understanding of RNA editing in adaptive immunity and identifies the ADAR2-MALT1-IL-17A axis as a potential therapeutic target for inflammatory conditions in the intestine.

immunology↗

bioRxiv2020

DDX5 targets tissue-specific RNAs to promote intestine tumorigenesis

Tumorigenesis in different segments of the intestinal tract involves tissue-specific oncogenic drivers. In the colon, complement component 3 (C3) activation is a major contributor to inflammation and malignancies. By contrast, tumorigenesis in the small intestine involves fatty acid-binding protein 1 (FABP1). However, little is known of the upstream mechanisms driving their expressions in different segments of the intestinal tract. Here, we report that an RNA binding protein DDX5 augments C3 and FABP1 expressions post-transcriptionally to promote tumorigenesis in the colon and small intestine, respectively. Mice with epithelial-specific knockout of DDX5 are protected from intestine tumorigenesis. The identification of DDX5 as the common upstream regulator of tissue-specific oncogenic molecules provides a new therapeutic target for intestine cancers.

cancer biology↗

bioRxiv2019

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins.

A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enabled mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale integrated analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the large-scale mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. Here we describe our analysis of 223 enhanced (eCLIP) datasets characterizing 150 RBPs in K562 and HepG2 cell lines, revealing a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multi-copy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation (enabling identification of branch points with single-nucleotide resolution) and provides genome-wide validation for a branch point-based scanning model for 3 splice site recognition. Further, we show that eCLIP peak co-occurrences across RBPs enables the discovery of novel co-interacting RBPs. Finally, we present a protocol for visualization of RBP:RNA complexes in the eCLIP workflow using biotin and standard chemiluminescent visualization reagents, enabling simplified confirmation of ribonucleoprotein enrichment without radioactivity. This work illustrates the value of integrated analysis across eCLIP profiling of RBPs with widely distinct functions to reveal novel RNA biology. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

genomics↗

bioRxiv2018

An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys

During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1kb) retained introns than they are in small retained introns or in non-retained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

molecular biology↗

bioRxiv2017

A Large-Scale Binding and Functional Map of Human RNA Binding Proteins

Genomes encompass all the information necessary to specify the development and function of an organism. In addition to genes, genomes also contain a myriad of functional elements that control various steps in gene expression. A major class of these elements function only when transcribed into RNA as they serve as the binding sites for RNA binding proteins (RBPs), which act to control post-transcriptional processes including splicing, cleavage and polyadenylation, RNA editing, RNA localization, stability, and translation. Despite the importance of these functional RNA elements encoded in the genome, they have been much less studied than genes and DNA elements. Here, we describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. These data expand the catalog of functional elements encoded in the human genome by addition of a large set of elements that function at the RNA level through interaction with RBPs.\n\nHighlightsO_LI223 eCLIP datasets for 150 RBPs reveal a wide variety of in vivo RNA target classes.\nC_LIO_LI472 knockdown/RNA-seq profiles of 263 RBPs reveal factor-responsive targets and integration with eCLIP indicates RNA expression and splicing regulatory patterns.\nC_LIO_LI78 RNA Bind-N-Seq profiles of in vitro binding motifs reveal links between in vitro and in vivo binding and indicate that eCLIP peaks that contain in vitro motifs are more strongly associated with regulation.\nC_LIO_LI274 maps of RBP subcellular localization by immunofluorescence indicate widespread organelle-specific RNA processing regulation.\nC_LIO_LI63 ChIP-seq profiles of DNA association suggest broad interconnectivity between chromatin association and RNA processing.\nC_LI

genomics↗