Publications by authors named "Kristen W Lynch"

63 Publications

Viral-induced alternative splicing of host genes promotes influenza replication.

Elife 2020 12 3;9. Epub 2020 Dec 3.

Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, United States.

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. Although 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, in several cases, specific inhibition of the IAV-induced splicing pattern 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 required for efficient splicing of the IAV M transcript in nuclear speckles. Finally, we find an increase in hnRNP K in nuclear speckles upon IAV infection, which may alter accessibility of hnRNP K for host transcripts thereby leading to a program of host splicing changes that promote IAV replication.
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http://dx.doi.org/10.7554/eLife.55500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735754PMC
December 2020

Alternative splicing and cancer: insights, opportunities, and challenges from an expanding view of the transcriptome.

Genes Dev 2020 08;34(15-16):1005-1016

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Over the past decade there has been increased awareness of the potential role of alternative splicing in the etiology of cancer. In particular, advances in RNA-Sequencing technology and analysis has led to a wave of discoveries in the last few years regarding the causes and functional relevance of alternative splicing in cancer. Here we discuss the current understanding of the connections between splicing and cancer, with a focus on the most recent findings. We also discuss remaining questions and challenges that must be addressed in order to use our knowledge of splicing to guide the diagnosis and treatment of cancer.
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http://dx.doi.org/10.1101/gad.338962.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397854PMC
August 2020

Meta-analysis of transcriptomic variation in T-cell populations reveals both variable and consistent signatures of gene expression and splicing.

RNA 2020 10 17;26(10):1320-1333. Epub 2020 Jun 17.

Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Human CD4 T cells are often subdivided into distinct subtypes, including Th1, Th2, Th17, and Treg cells, that are thought to carry out distinct functions in the body. Typically, these T-cell subpopulations are defined by the expression of distinct gene repertoires; however, there is variability between studies regarding the methods used for isolation and the markers used to define each T-cell subtype. Therefore, how reliably studies can be compared to one another remains an open question. Moreover, previous analysis of gene expression in CD4 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 determine the consistency in gene expression and splicing within each subtype across studies. 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 appear to consistently differentiate between subsets, including a few Treg-specific splicing patterns. Together this work highlights the heterogeneity in gene expression between samples designated as the same subtype, but also suggests additional markers that can be used to define functional groupings.
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http://dx.doi.org/10.1261/rna.075929.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491319PMC
October 2020

Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells.

Nucleic Acids Res 2020 06;48(10):5710-5719

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.
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http://dx.doi.org/10.1093/nar/gkaa295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261192PMC
June 2020

Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing.

Genome Res 2019 12 14;29(12):2046-2055. Epub 2019 Nov 14.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.
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http://dx.doi.org/10.1101/gr.248435.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886501PMC
December 2019

RNA Binding Protein CELF2 Regulates Signal-Induced Alternative Polyadenylation by Competing with Enhancers of the Polyadenylation Machinery.

Cell Rep 2019 09;28(11):2795-2806.e3

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The 3' UTR (UTR) of human mRNAs plays a critical role in controlling protein expression and function. Importantly, 3' UTRs of human messages are not invariant for each gene but rather are shaped by alternative polyadenylation (APA) in a cell state-dependent manner, including in response to T cell activation. However, the proteins and mechanisms driving APA regulation remain poorly understood. Here we show that the RNA-binding protein CELF2 controls APA of its own message in a signal-dependent manner by competing with core enhancers of the polyadenylation machinery for binding to RNA. We further show that CELF2 binding overlaps with APA enhancers transcriptome-wide, and almost half of 3' UTRs that undergo T cell signaling-induced APA are regulated in a CELF2-dependent manner. These studies thus reveal CELF2 to be a critical regulator of 3' UTR identity in T cells and demonstrate an additional mechanism for CELF2 in regulating polyadenylation site choice.
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http://dx.doi.org/10.1016/j.celrep.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752737PMC
September 2019

Functional and Mechanistic Interplay of Host and Viral Alternative Splicing Regulation during Influenza Infection.

Cold Spring Harb Symp Quant Biol 2019 ;84:123-131

Department of Biochemistry and Biophysics Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Alternative splicing is a pervasive gene regulatory mechanism utilized by both mammalian cells and viruses to expand their genomic coding capacity. The process of splicing and the RNA sequences that guide this process are the same in mammalian and viral transcripts; however, viruses lack the splicing machinery and therefore must usurp both the host spliceosome and many of the associated regulatory proteins in order to correctly process their genes. Here, we use the example of the influenza A virus to both describe how viruses utilize host splicing factors to regulate their own splicing and provide examples of how viral infection can, in turn, alter host splicing. Importantly, we show that at least some of the viral-induced changes in host splicing occur in genes that alter the efficiency of influenza replication. We emphasize the importance of increased understanding of the mechanistic interplay between host and viral splicing, and its functional consequences, in uncovering potential antiviral vulnerabilities.
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http://dx.doi.org/10.1101/sqb.2019.84.039040DOI Listing
January 2019

Structural-functional interactions of NS1-BP protein with the splicing and mRNA export machineries for viral and host gene expression.

Proc Natl Acad Sci U S A 2018 12 11;115(52):E12218-E12227. Epub 2018 Dec 11.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390;

The influenza virulence factor NS1 protein interacts with the cellular NS1-BP protein to promote splicing and nuclear export of the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix protein and is alternatively spliced into the M2 mRNA, which is translated into the M2 ion channel. These proteins have key functions in viral trafficking and budding. To uncover the NS1-BP structural and functional activities in splicing and nuclear export, we performed proteomics analysis of nuclear NS1-BP binding partners and showed its interaction with constituents of the splicing and mRNA export machineries. NS1-BP BTB domains form dimers in the crystal. Full-length NS1-BP is a dimer in solution and forms at least a dimer in cells. Mutations suggest that dimerization is important for splicing. The central BACK domain of NS1-BP interacts directly with splicing factors such as hnRNP K and PTBP1 and with the viral NS1 protein. The BACK domain is also the site for interactions with mRNA export factor Aly/REF and is required for viral M mRNA nuclear export. The crystal structure of the C-terminal Kelch domain shows that it forms a β-propeller fold, which is required for the splicing function of NS1-BP. This domain interacts with the polymerase II C-terminal domain and SART1, which are involved in recruitment of splicing factors and spliceosome assembly, respectively. NS1-BP functions are not only critical for processing a subset of viral mRNAs but also impact levels and nuclear export of a subset of cellular mRNAs encoding factors involved in metastasis and immunity.
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http://dx.doi.org/10.1073/pnas.1818012115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310826PMC
December 2018

Aberrant splicing in B-cell acute lymphoblastic leukemia.

Nucleic Acids Res 2018 11;46(21):11357-11369

Department of Pathology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

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 these samples 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 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, 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 contributes to disease pathogenesis.
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http://dx.doi.org/10.1093/nar/gky946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277088PMC
November 2018

Alternative pre-mRNA splicing switch controls hESC pluripotency and differentiation.

Genes Dev 2018 09;32(17-18):1103-1104

Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Alternative splicing (AS) of pre-mRNAs is a ubiquitous process in mammals that is tightly regulated in a cell type- and cell state-dependent manner. However, the details of how splicing is regulated to impact specific cell fate decisions remains incompletely understood. A study by Yamazaki and colleagues (pp. 1161-1174) in this issue of provides exciting new insight into the role and regulation of splicing in the maintenance of pluripotency of human embryonic stem cells (hESCs). In brief, they show that AS of several genes is robustly regulated upon differentiation of hESCs. One of these genes, T-cell factor 3 (), is regulated at least in part through the activity of heterogeneous nuclear ribonucleoproteins H1 and F (hnRNP H/F) to control the mutually exclusive expression of the encoded E12 and E47 transcription regulators. The investigators demonstrate that reduced expression of hnRNP H/F favors expression of E47, which in turn decreases E-cadherin expression to promote hESC differentiation. In contrast, high levels of hnRNP H/F induce expression of E12 to maintain pluripotency. Thus, this work provides at least one new link between AS and control of human stem cell fate and suggests a broader role of splicing in pluripotency.
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http://dx.doi.org/10.1101/gad.318451.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120719PMC
September 2018

Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing.

Nat Commun 2018 06 19;9(1):2407. Epub 2018 Jun 19.

Departments of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 422 Curie Blvd, Philadelphia, PA, 19104, USA.

Three of the eight RNA segments encoded by the influenza A virus (IAV) undergo alternative splicing to generate distinct proteins. Previously, we found that host proteins hnRNP K and NS1-BP regulate IAV M segment splicing, but the mechanistic details were unknown. Here we show NS1-BP and hnRNP K bind M mRNA downstream of the M2 5' splice site (5'ss). NS1-BP binds most proximal to the 5'ss, partially overlapping the U1 snRNP binding site, while hnRNP K binds further downstream and promotes U1 snRNP recruitment. Mutation of either or both the hnRNP K and NS1-BP-binding sites results in M segment mis-splicing and attenuated IAV replication. Additionally, we show that hnRNP K and NS1-BP regulate host splicing events and that viral infection causes mis-splicing of some of these transcripts. Therefore, our proposed mechanism of hnRNP K/NS1-BP mediated IAV M splicing provides potential targets of antiviral intervention and reveals novel host functions for these proteins.
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http://dx.doi.org/10.1038/s41467-018-04779-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008300PMC
June 2018

HnRNP L represses cryptic exons.

RNA 2018 06 26;24(6):761-768. Epub 2018 Mar 26.

Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.

The fidelity of RNA splicing is regulated by a network of splicing enhancers and repressors, although the rules that govern this process are not yet fully understood. One mechanism that contributes to splicing fidelity is the repression of nonconserved cryptic exons by splicing factors that recognize dinucleotide repeats. We previously identified that TDP-43 and PTBP1/PTBP2 are capable of repressing cryptic exons utilizing UG and CU repeats, respectively. Here we demonstrate that hnRNP L () also represses cryptic exons by utilizing exonic CA repeats, particularly near the 5'SS. We hypothesize that hnRNP L regulates CA repeat repression for both cryptic exon repression and developmental processes such as T cell differentiation.
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http://dx.doi.org/10.1261/rna.065508.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959245PMC
June 2018

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing.

J Biol Chem 2017 11 15;292(44):18240-18255. Epub 2017 Sep 15.

From the Pharmacology Graduate Group,

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and knock-out ( DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins RNA-Seq of WT and DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.
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http://dx.doi.org/10.1074/jbc.M117.813527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672046PMC
November 2017

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes.

Genome Res 2017 08 16;27(8):1360-1370. Epub 2017 May 16.

Department of Genetics.

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 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 coregulation 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 coregulated 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 suggests this antagonism is conserved throughout evolution.
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http://dx.doi.org/10.1101/gr.220517.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538552PMC
August 2017

Influenza virus mRNA trafficking through host nuclear speckles.

Nat Microbiol 2016 05 27;1(7):16069. Epub 2016 May 27.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA.

Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.
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http://dx.doi.org/10.1038/nmicrobiol.2016.69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917225PMC
May 2016

Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells.

RNA Biol 2016 06 20;13(6):569-81. Epub 2016 Apr 20.

a Department of Biochemistry and Biophysics , University of Pennsylvania Perelman School of Medicine , Philadelphia , PA , USA.

CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts.
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http://dx.doi.org/10.1080/15476286.2016.1176663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962813PMC
June 2016

A new view of transcriptome complexity and regulation through the lens of local splicing variations.

Elife 2016 Feb 1;5:e11752. Epub 2016 Feb 1.

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.
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http://dx.doi.org/10.7554/eLife.11752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801060PMC
February 2016

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

Cancer Discov 2015 Dec 29;5(12):1282-95. Epub 2015 Oct 29.

Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Immunology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Cell & Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Unlabelled: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.

Significance: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
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http://dx.doi.org/10.1158/2159-8290.CD-15-1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670800PMC
December 2015

Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation.

Genes Dev 2015 Oct;29(19):2054-66

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennysylvania 19104, USA.

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.
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http://dx.doi.org/10.1101/gad.267245.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604346PMC
October 2015

Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons.

RNA 2015 Dec 5;21(12):2053-66. Epub 2015 Oct 5.

Department of Biochemistry and Biophysics Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

HnRNP L is a ubiquitous splicing-regulatory protein that is critical for the development and function of mammalian T cells. Previous work has identified a few targets of hnRNP L-dependent alternative splicing in T cells and has described transcriptome-wide association of hnRNP L with RNA. However, a comprehensive analysis of the impact of hnRNP L on mRNA expression remains lacking. Here we use next-generation sequencing to identify transcriptome changes upon depletion of hnRNP L in a model T-cell line. We demonstrate that hnRNP L primarily regulates cassette-type alternative splicing, with minimal impact of hnRNP L depletion on transcript abundance, intron retention, or other modes of alternative splicing. Strikingly, we find that binding of hnRNP L within or flanking an exon largely correlates with exon repression by hnRNP L. In contrast, exons that are enhanced by hnRNP L generally lack proximal hnRNP L binding. Notably, these hnRNP L-enhanced exons share sequence and context features that correlate with poor nucleosome positioning, suggesting that hnRNP may enhance inclusion of a subset of exons via a cotranscriptional or epigenetic mechanism. Our data demonstrate that hnRNP L controls inclusion of a broad spectrum of alternative cassette exons in T cells and suggest both direct RNA regulation as well as indirect mechanisms sensitive to the epigenetic landscape.
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http://dx.doi.org/10.1261/rna.052969.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647460PMC
December 2015

TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells.

Nucleic Acids Res 2015 Oct 10;43(18):9006-16. Epub 2015 Aug 10.

Department of Biochemistry and Biophysics Perelman, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells.
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http://dx.doi.org/10.1093/nar/gkv816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605305PMC
October 2015

Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling.

Proc Natl Acad Sci U S A 2015 Apr 13;112(17):E2139-48. Epub 2015 Apr 13.

Departments of Biochemistry and Biophysics and Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and

Studies in several cell types have highlighted dramatic and diverse changes in mRNA processing that occur upon cellular stimulation. However, the mechanisms and pathways that lead to regulated changes in mRNA processing remain poorly understood. Here we demonstrate that expression of the splicing factor CELF2 (CUGBP, Elav-like family member 2) is regulated in response to T-cell signaling through combined increases in transcription and mRNA stability. Transcriptional induction occurs within 6 h of stimulation and is dependent on activation of NF-κB. Subsequently, there is an increase in the stability of the CELF2 mRNA that correlates with a change in CELF2 3'UTR length and contributes to the total signal-induced enhancement of CELF2 expression. Importantly, we uncover dozens of splicing events in cultured T cells whose changes upon stimulation are dependent on CELF2 expression, and provide evidence that CELF2 controls a similar proportion of splicing events during human thymic T-cell development. Taken together, these findings expand the physiologic impact of CELF2 beyond that previously documented in developing neuronal and muscle cells to T-cell development and function, identify unappreciated instances of alternative splicing in the human thymus, and uncover novel mechanisms for CELF2 regulation that may broadly impact CELF2 expression across diverse cell types.
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http://dx.doi.org/10.1073/pnas.1423695112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418860PMC
April 2015

PSF: nuclear busy-body or nuclear facilitator?

Wiley Interdiscip Rev RNA 2015 Jul-Aug;6(4):351-67. Epub 2015 Apr 1.

Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA.

PTB-associated splicing factor (PSF) is an abundant and essential nucleic acid-binding protein that participates in a wide range of gene regulatory processes and cellular response pathways. At the protein level, PSF consists of multiple domains, many of which remain poorly characterized. Although grouped in a family with the proteins p54nrb/NONO and PSPC1 based on sequence homology, PSF contains additional protein sequence not included in other family members. Consistently, PSF has also been implicated in functions not ascribed to p54nrb/NONO or PSPC1. Here, we provide a review of the cellular activities in which PSF has been implicated and what is known regarding the mechanisms by which PSF functions in each case. We propose that the complex domain arrangement of PSF allows for its diversity of function and integration of activities. Finally, we discuss recent evidence that individual activities of PSF can be regulated independently from one another through the activity of domain-specific co-factors.
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http://dx.doi.org/10.1002/wrna.1280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478221PMC
March 2016

Thoughts on NGS, alternative splicing and what we still need to know.

Authors:
Kristen W Lynch

RNA 2015 Apr;21(4):683-4

Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104-6059, USA

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http://dx.doi.org/10.1261/rna.050419.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371332PMC
April 2015

Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense.

Cell 2014 Aug;158(4):764-777

Department of Microbiology, Penn Genome Frontiers Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.
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http://dx.doi.org/10.1016/j.cell.2014.06.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134512PMC
August 2014

Cell-based splicing of minigenes.

Methods Mol Biol 2014 ;1126:243-55

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Cell-based splicing of minigenes is used extensively in the analysis of alternative splicing events. In particular, such assays are critical for identifying or confirming the in vivo relevance of cis- and trans-acting factors in the regulation of particular splicing patterns. Here we provide detailed information on the methods specific to the cell-based analysis of minigene splicing. In addition, we discuss some of the theoretical considerations that must be given to the design of the minigene and subsequent experimental conditions.
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http://dx.doi.org/10.1007/978-1-62703-980-2_18DOI Listing
October 2014

Mechanisms of spliceosomal assembly.

Methods Mol Biol 2014 ;1126:35-43

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Pre-mRNA splicing is a key step for generating mature protein-coding mRNA. An RNA-protein complex known as the spliceosome carries out the chemistry of pre-mRNA splicing. However, several pre-spliceosomal intermediates are assembled on the pre-mRNA before the formation of the catalytically activated spliceosome. The progression to the activated spliceosome involves a cascade of the rearrangement events of the RNA-RNA, RNA-protein, and protein-protein interactions within the pre-spliceosomal intermediates. These rearrangements generate multiple combinatorial interactions of the spliceosome with the substrate, which enhances the accuracy of the splice site selection. Each rearrangement also represents a step at which splicing can potentially be subjected to regulation. The aim of this chapter is to provide an overview of the components of the spliceosome and their rearrangements along the spliceosome assembly pathway.
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http://dx.doi.org/10.1007/978-1-62703-980-2_3DOI Listing
October 2014

An optogenetic gene expression system with rapid activation and deactivation kinetics.

Nat Chem Biol 2014 Mar 12;10(3):196-202. Epub 2014 Jan 12.

1] Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA. [2] Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA. [3] Structural Biology Initiative, Advanced Science Research Center, City University of New York, New York, New York, USA.

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.
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http://dx.doi.org/10.1038/nchembio.1430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3944926PMC
March 2014