Biology subjects

Lynch, K. W.

Publications and source records attributed to Lynch, K. W..

17 recordsLinked to original sources

bioRxiv2025

Coordination of nuclear RNA processing by speckle-localized kinase TAOK2

Nuclear speckles are membraneless organelles that act as active splicing hubs especially at sites of high transcription. Emerging views of this dynamic subnuclear structure place it as a hub of RNA processing, impacting steps from transcription to export. To manage this complex microcosm of RNA metabolism, nuclear speckles also require kinases and phosphorylation to execute their functions. The nuclear speckle-localized kinase, TAOK2, mediates the splicing and export of viral transcripts at the nuclear speckle, but its role in the processing of cellular transcripts was unknown. We used siRNA knockdown of TAOK2 and assessed RNA transcripts in both whole cell and nucleocytoplasmic fractions to characterize the complete endogenous effects of TAOK2. We found that TAOK2 knockdown impacts over 10% of the transcriptome, through changes in alternative splicing, export and transcript abundance. Cellular and biochemical phosphoproteomics further revealed nuclear speckle scaffolding proteins SRRM1 and SRRM2 as potential direct phosphorylation targets of TAOK2, mediating its large effects on speckle integrity and speckle-localized splicing. Indeed, knockdown of TAOK2 perturbs almost all speckle-resident serine/arginine (SR)-rich proteins while leaving heterogeneous ribonucleoproteins unperturbed. Altogether, we propose that phosphorylation of SRRM1/2 by TAOK2 plays a structural maintenance role that impacts SR protein-driven exon inclusion at the nuclear speckle.

molecular biology↗

bioRxiv2025

Nonsense-mediated decay controls a negative feedback loop in innate immune sensing

Nonsense-mediated decay (NMD) is an mRNA decay pathway which degrades potential harmful transcripts that contain premature termination codons. However, NMDs importance also extends to the control of isoform abundance under physiological conditions. During viral infection, NMD is inhibited through numerous mechanisms; however, NMD has been shown to have both antiviral as well as proviral activities, raising further questions into the role and control of NMD during viral infection. These observations have led us to investigate the potential involvement of NMD in dsRNA sensing as a mechanism that might explain these discrepancies. Using EIF4A2 exon 10B inclusion as an example of AS-NMD isoform accumulating during viral infection, we show that dsRNA sensing inhibits NMD. This effect is correlated with translational blockade and is driven primarily by RNaseL activation, and by PKR in the absence of RNaseL activation. Surprisingly, NMD inhibition limits the induction of IFN-{beta} as well as interferon-stimulated genes, and this effect is upstream of IRF3 phosphorylation and translocation to the nucleus. NMD inhibition also decreases PKR and RNaseL activation as well as PIC-mediated cell death by decreasing the dsRNA content, suggesting NMD directly controls dsRNA sensing by controlling the dsRNA load. Therefore, inhibition of NMD upon dsRNA sensing provides a negative feedback loop that contributes to shaping the innate immune sensing pathways. SIGNIFICANCENonsense-mediated decay (NMD) is a translation-dependent mRNA decay pathway that plays an important role in shaping the transcriptome. In this manuscript, we show that dsRNA sensing, as is typical during viral infection, inhibits NMD mainly through the translational inhibition caused by RNaseL activation. This NMD inhibition forms a negative-feedback loop that limits dsRNA sensing, thus preventing overactivation of dsRNA-mediated pathways. These findings contribute to a better understanding of the molecular mechanisms that limit antiviral responses as well as inflammation and inform the critical role that mRNA processes plays in innate immunity.

immunology↗

bioRxiv2025

Integrative analysis of RNA binding proteins identifies DDX55 as a novel regulator of 3'UTR isoform diversity

The 3 untranslated regions (3UTRs) of mRNAs play a critical role in controlling gene expression and function because they contain binding sites for microRNAs and RNA binding proteins (RBPs) that alter mRNA stability, localization, and translation. Most mRNA 3 ends contain multiple polyadenylation sites (PAS) that can be utilized in condition-specific manners, a process known as alternative polyadenylation (APA), however the mechanisms driving the regulation of APA remain poorly characterized. By integrating a large set of over 500 RNA binding protein (RBP) depletion and binding experiments across two cell lines generated by the ENCODE consortium, we uncovered a number of RBPs in each cell type whose depletion leads to widespread alteration of 3 UTR patterns. These include not only known regulators of APA, but also many putative novel regulators of 3UTR isoform expression. We focused analysis on the largely unstudied DEAD box RNA helicase, DDX55, and validate its novel role in 3UTR isoform regulation using molecular assays and targeted 3 end sequencing experiments. Our findings identify DDX55 as a new regulator of APA, particularly at PAS that contain features of RNA secondary structure. Our data also suggest additional previously unrecognized regulators of 3 UTR processing and differential stability.

genomics↗

bioRxiv2025

Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

Interferon Regulatory Factor 7 (IRF7), and its homologue IRF3, are master transcriptional regulators of the innate immune response. IRF7 binds to promoters of interferon {beta} (IFN{beta}) and several IFNs as a homodimer or as a heterodimer with IRF3 to drive expression of these type I IFNs, which in turn activate downstream signaling pathways to promote expression of antiviral genes. Here we demonstrate that alternative splicing of the first intron within the coding region of IRF7 is highly regulated across immune tissues and in response to immunologic signals including viral infection. Retention of this intron generates an alternative translation start site, resulting in a N-terminally extended form of the protein (exIRF7) with distinct function from the canonical version of IRF7 (cIRF7). We find that exIRF7 uniquely activates a gene expression program, including IFN{beta}, in response to innate immune triggers. Mechanistically, this enhanced activity of exIRF7 relative to cIRF7 is through increased homodimerization and association with IRF3 on DNA. Furthermore, the enhanced transcriptional activity of exIRF7 controls viral infection to a greater extent than cIRF7, demonstrating that alternative splicing of IRF7 is a previously unrecognized mechanism used by cells to tune the interferon response to control viral infections and other immune challenges. HighlightsO_LIIntron retention in the human IRF7 gene generates a distinct protein isoform that differs in the N-terminus. C_LIO_LIIRF7 intron retention is regulated in a stimuli- and cell-type specific manner. C_LIO_LIThe extended version of IRF7, produced by intron retention, exhibits enhanced transcriptional activation of type I interferon genes. C_LIO_LICells expressing the extended version of IRF7 are more resistant to viral infection. C_LI

molecular biology↗

bioRxiv2024

Machine learning-optimized targeted detection of alternative splicing

RNA-sequencing (RNA-seq) is widely adopted for transcriptome analysis but has inherent biases which hinder the comprehensive detection and quantification of alternative splicing. To address this, we present an efficient targeted RNA-seq method that greatly enriches for splicing-informative junction-spanning reads. Local Splicing Variation sequencing (LSV-seq) utilizes multiplexed reverse transcription from highly scalable pools of primers anchored near splicing events of interest. Primers are designed using Optimal Prime, a novel machine learning algorithm trained on the performance of thousands of primer sequences. In experimental benchmarks, LSV-seq achieves high on-target capture rates and concordance with RNA-seq, while requiring significantly lower sequencing depth. Leveraging deep learning splicing code predictions, we used LSV-seq to target events with low coverage in GTEx RNA-seq data and newly discover hundreds of tissue-specific splicing events. Our results demonstrate the ability of LSV-seq to quantify splicing of events of interest at high-throughput and with exceptional sensitivity.

genomics↗

bioRxiv2023

Targeting mitophagy in SRSF2 mutant hematologic malignancies

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

cancer biology↗

bioRxiv2022

Alternative Splicing of Apoptosis Genes Promotes Human T Cell Survival

Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.

molecular biology↗

bioRxiv2022

RNase L-mediated RNA decay alters 3' end formation and splicing of host mRNAs

The antiviral endoribonuclease, RNase L, is a vital component of the mammalian innate immune response that destroys host and viral RNA to reduce viral gene expression. Herein, we show that a consequence of RNase L-mediated decay of cytoplasmic host RNAs is the widespread re-localization of RNA-binding proteins (RBPs) from the cytoplasm to the nucleus, due to the presence of nuclear RNA. Concurrently, we observe global alterations to host RNA processing in the nucleus, including alterations of splicing and 3 end formation, with the latter leading to downstream of gene (DoG) transcripts. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and coincide with the retention of their mRNAs in the nucleus. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is fundamentally dictated by the availability of RNA in each compartment and thus viral infections that trigger cytoplasmic RNA degradation alter RNA processing due to the nuclear influx of RNA binding proteins.

immunology↗

bioRxiv2021

Identifying common transcriptome signatures of cancer by interpreting deep learning models

Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified, signifying that cancer cases display common hallmark molecular features. It is not clear however whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types. Here, in order to agnostically identify transcriptomic features that are commonly shared between cancer types, we used RNA-Seq datasets encompassing thousands of samples from 19 healthy tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression or splice junction use, to distinguish between healthy and tumor samples. All three models achieve high precision, recall and accuracy on test sets derived from 13 datasets used during training and on an independent test dataset, indicating that our models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints, suggesting that they have important cellular functions. Importantly, we found that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer. Finally, our results also highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features across a large array of solid tumor types. The transcriptomic features that we highlight here define cancer signatures that may reflect causal variations or consequences of disease state, or a combination of both.

cancer biology↗

bioRxiv2021

PRMT5 promotes symmetric dimethylation of RNA processing proteins and modulates activated T cell alternative splicing and Ca2+/NFAT signaling

Protein Arginine Methyltransferase (PRMT) 5 is the major type 2 methyltransferase catalyzing symmetric dimethylation (SDM) of arginine. PRMT5 inhibition or deletion in CD4 Th cells reduces TcR engagement-induced IL-2 production and Th cell expansion and confers protection against experimental autoimmune encephalomyelitis (EAE), the animal model of Multiple Sclerosis. However, the mechanisms by which PRMT5 modulates T helper (Th) cell proliferation are still not completely understood and neither are the methylation targets in T cells. In this manuscript, we uncover the role of PRMT5 on alternative splicing (AS) in activated T cells and identify several targets of PRMT5 SDM involved in splicing. In addition, we find a possible link between PRMT5 mediated AS of Trpm4 (Transient Receptor Potential Cation Channel Subfamily M Member 4) and TcR/NFAT signaling/IL-2 production. This understanding may guide development of drugs targeting these processes to benefit patients with T cell-mediated diseases.

immunology↗

bioRxiv2020

MOCCASIN: A method for correcting for known and unknown confounders in RNA splicing analysis

While the effects of confounders on gene expression analysis have been extensively studied there is a lack of equivalent analysis and tools for RNA splicing analysis. Here we assess the effect of confounders in two large public RNA-Seq datasets (TARGET, ENCODE), develop a new method, MOCCASIN, to correct the effect of both known and unknown confounders on RNA splicing quantification, and demonstrate MOCCASINs effectiveness on both synthetic and real data.

genomics↗

bioRxiv2020

Viral-Induced Alternative Splicing of Host Genes Promotes Influenza Replication

Viral infection induces the expression of numerous host genes that impact the outcome of infection. Here we show that infection of human lung epithelial cells with Influenza A virus (IAV) also induces a broad program of alternative splicing of host genes. While these splicing-regulated genes are not enriched for canonical regulators of viral infection, we find that many of these genes do impact replication of IAV. Moreover, specific inhibition of the IAV-induced splicing in several cases also attenuates viral infection. We further show that approximately a quarter of the IAV-induced splicing events are regulated by hnRNP K, a host protein that required for efficient splicing of the IAV M transcript in nuclear speckles. Notably, we find that hnRNP K accumulates in nuclear speckles upon IAV infection, which is likely to alter the accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.

molecular biology↗

bioRxiv2020

Alternative splicing redefines landscape of commonly mutated genes in acute myeloid leukemia

Most genes associated with Acute Myeloid Leukemia (AML) are mutated in less than 10% of patients, suggesting alternative mechanisms for gene disruption contribute to this disease. Here we find a set of splicing events that disrupt the expression of a subset of AML-associated genes, including EZH2 and ZRSR2, independent of known somatic mutations. Most strikingly, in at least one cohort, aberrant splicing triples the number of patients with a reduction in functional EZH2 as compared to that predicted by somatic mutation of EZH2 alone. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlights the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

genomics↗

bioRxiv2019

Set2-mediated alternative splicing of Srsf11 regulates cocaine reward behavior

Alternative splicing is a key mechanism for neuronal gene regulation, and is grossly altered in mouse brain reward regions following investigator-administered cocaine. It is well established that cocaine epigenetically regulates transcription, yet mechanism(s) by which cocaine-induced epigenetic modifications regulate alternative splicing is largely unexplored. Our group and others have previously identified the histone modification, H3K36me3, as a putative splicing regulator. However, it has not yet been possible to establish the direct causal relevance of this modification to alternative splicing in brain or any other context. We found that mouse cocaine self-administration caused widespread alternative splicing, concomitant with enrichment of H3K36me3 at splice junctions. Differentially spliced genes were enriched in the motif for splice factor, Srsf11, which was both differentially spliced and enriched in H3K36me3. Epigenetic editing led us to conclude that H3K36me3 functions directly in alternative splicing of Srsf11, and that Set2 mediated H3K36me3 bidirectionally regulates cocaine intake.

neuroscience↗

bioRxiv2019

Meta-Analysis of Transcriptomic Variation in T cell Populations Reveals Novel Signatures of Gene Expression and Splicing

Distinct T cell subtypes are typically defined by the expression of distinct gene repertoires. However, there is variability between studies regarding the markers used to define each T cell subtype. Moreover, previous analysis of gene expression in T cell subsets has largely focused on gene expression rather than alternative splicing. Here we take a meta-analysis approach, comparing eleven independent RNA-Seq studies of human Th1, Th2, Th17 and/or Treg cells to identify transcriptomic features that correlate consistently with subtype. We find that known master-regulators are consistently enriched in the appropriate subtype, however, cytokines and other genes often used as markers are more variable. Importantly, we also identify previously unknown transcriptomic markers that consistently differentiate between subsets, including a few Treg-specific splicing patterns. Together this work highlights the heterogeneity in gene expression between isolates of the same subtype, but also suggests additional markers that can be used to define functional groupings.

immunology↗

bioRxiv2017

Aberrant splicing in B-cell acute lymphoblastic leukemia

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing them to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ~100 SFs, e.g. hnRNPA1. hnRNPA1 3UTR was pervasively misspliced, yielding the transcript subject to nonsense-mediated decay. Thus, we knocked it down in B-lymphoblastoid cells, identified 213 hnRNPA1-dependent splicing events, and defined the hnRNPA1 splicing signature in pediatric leukemias. One of its elements was DICER1, a known tumor suppressor gene; its LSVs were consistent with reduced translation of DICER1 mRNA. Additionally, we searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 genes. 77 LSVs were confirmed using two large independent B-ALL RNA-seq datasets. In fact, the twenty most common B-ALL drivers showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contribute to disease pathogenesis.

cancer biology↗

bioRxiv2017

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors. We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic co-regulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these co-regulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, and Drosophila suggests this antagonism is conserved through evolution.

genomics↗