Publications by authors named "Michael J Mallory"

27 Publications

  • Page 1 of 1

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

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

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

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

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

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

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

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

Acetylation of the transcriptional repressor Ume6p allows efficient promoter release and timely induction of the meiotic transient transcription program in yeast.

Mol Cell Biol 2014 Feb 2;34(4):631-42. Epub 2013 Dec 2.

Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Stratford, New Jersey, USA.

Differentiation programs require strict spatial and temporal control of gene transcription. Genes expressed during meiotic development in Saccharomyces cerevisiae display transient induction and repression. Early meiotic gene (EMG) repression during mitosis is achieved by recruiting both histone deacetylase and chromatin remodeling complexes to their promoters by the zinc cluster DNA binding protein Ume6p. Ume6p repression is relieved by ubiquitin-mediated destruction that is stimulated by Gcn5p-induced acetylation. In this report, we demonstrate that Gcn5p acetylation of separate lysines within the zinc cluster domain negatively impacts Ume6p DNA binding. Mimicking lysine acetylation using glutamine substitution mutations decreased Ume6p binding efficiency and resulted in partial derepression of Ume6p-regulated genes. Consistent with this result, molecular modeling predicted that these lysine side chains are adjacent to the DNA phosphate backbone, suggesting that acetylation inhibits Ume6p binding by electrostatic repulsion. Preventing acetylation did not impact final EMG induction levels during meiosis. However, a delay in EMG induction was observed, which became more severe in later expression classes, ultimately resulting in delayed and reduced execution of the meiotic nuclear divisions. These results indicate that Ume6p acetylation ensures the proper timing of the transient transcription program during meiotic development.
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http://dx.doi.org/10.1128/MCB.00256-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911482PMC
February 2014

Paralogs hnRNP L and hnRNP LL exhibit overlapping but distinct RNA binding constraints.

PLoS One 2013 11;8(11):e80701. Epub 2013 Nov 11.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

HnRNP (heterogeneous nuclear ribonucleoprotein) proteins are a large family of RNA-binding proteins that regulate numerous aspects of RNA processing. Interestingly, several paralogous pairs of hnRNPs exist that exhibit similar RNA-binding specificity to one another, yet have non-redundant functional targets in vivo. In this study we systematically investigate the possibility that the paralogs hnRNP L and hnRNP LL have distinct RNA binding determinants that may underlie their lack of functional redundancy. Using a combination of RNAcompete and native gel analysis we find that while both hnRNP L and hnRNP LL preferentially bind sequences that contain repeated CA dinucleotides, these proteins differ in their requirement for the spacing of the CAs. Specifically, hnRNP LL has a more stringent requirement for a two nucleotide space between CA repeats than does hnRNP L, resulting in hnRNP L binding more promiscuously than does hnRNP LL. Importantly, this differential requirement for the spacing of CA dinucleotides explains the previously observed differences in the sensitivity of hnRNP L and LL to mutations within the CD45 gene. We suggest that overlapping but divergent RNA-binding preferences, as we show here for hnRNP L and hnRNP LL, may be commonplace among other hnRNP paralogs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080701PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823766PMC
February 2015

Transcriptome-wide RNA interaction profiling reveals physical and functional targets of hnRNP L in human T cells.

Mol Cell Biol 2014 Jan 28;34(1):71-83. Epub 2013 Oct 28.

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

The RNA processing factor hnRNP L is required for T cell development and function. However, the spectrum of direct targets of hnRNP L activity in T cells has yet to be defined. In this study, we used cross-linking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq) to identify the RNA binding sites of hnRNP L within the transcriptomes of human CD4(+) and cultured Jurkat T cells. We find that hnRNP L binds preferentially to transcripts encoding proteins involved in RNA processing and in Wnt and T cell receptor (TCR) signaling. This binding is largely conserved across both quiescent and activated T cells, in agreement with the critical role of hnRNP L throughout T cell biology. Importantly, based on the binding profile of hnRNP L, we validate numerous instances of hnRNP L-dependent alternative splicing of genes critical to T cell function. We further show that alternative exons with weak 5' splice site sequences specifically show a strong correlation between hnRNP L binding and hnRNP L-dependent splicing regulation. Together, these data provide the first transcriptome-wide analysis of the RNA targets of hnRNP L in lymphoid cells and add to the functional understanding of hnRNP L in human biology.
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http://dx.doi.org/10.1128/MCB.00740-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911283PMC
January 2014

Mutually dependent degradation of Ama1p and Cdc20p terminates APC/C ubiquitin ligase activity at the completion of meiotic development in yeast.

Cell Div 2013 Jul 1;8(1). Epub 2013 Jul 1.

Department of Molecular Biology, UMDNJ-SOM, 2 Medical Center Drive,, , 08084, USA.

Background: The execution of meiotic nuclear divisions in S. cerevisiae is regulated by protein degradation mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The correct timing of APC/C activity is essential for normal chromosome segregation. During meiosis, the APC/C is activated by the association of either Cdc20p or the meiosis-specific factor Ama1p. Both Ama1p and Cdc20p are targeted for degradation as cells exit meiosis II with Cdc20p being destroyed by APC/CAma1. In this study we investigated how Ama1p is down regulated at the completion of meiosis.

Findings: Here we show that Ama1p is a substrate of APC/CCdc20 but not APC/CCdh1 in meiotic cells. Cdc20p binds Ama1p in vivo and APC/CCdc20 ubiquitylates Ama1p in vitro. Ama1p ubiquitylation requires one of two degradation motifs, a D-box and a "KEN-box" like motif called GxEN. Finally, Ama1p degradation does not require its association with the APC/C via its conserved APC/C binding motifs (C-box and IR) and occurs simultaneously with APC/CAma1-mediated Cdc20p degradation.

Conclusions: Unlike the cyclical nature of mitotic cell division, meiosis is a linear pathway leading to the production of quiescent spores. This raises the question of how the APC/C is reset prior to spore germination. This and a previous study revealed that Cdc20p and Ama1p direct each others degradation via APC/C-dependent degradation. These findings suggest a model that the APC/C is inactivated by mutual degradation of the activators. In addition, these results support a model in which Ama1p and Cdc20p relocate to the substrate address within the APC/C cavity prior to degradation.
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http://dx.doi.org/10.1186/1747-1028-8-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734102PMC
July 2013

Gcn5p-dependent acetylation induces degradation of the meiotic transcriptional repressor Ume6p.

Mol Biol Cell 2012 May 21;23(9):1609-17. Epub 2012 Mar 21.

Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ 08084, USA.

Ume6p represses early meiotic gene transcription in Saccharomyces cerevisiae by recruiting the Rpd3p histone deacetylase and chromatin-remodeling proteins. Ume6p repression is relieved in a two-step destruction process mediated by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. The first step induces partial Ume6p degradation when vegetative cells shift from glucose- to acetate-based medium. Complete proteolysis happens only upon meiotic entry. Here we demonstrate that the first step in Ume6p destruction is controlled by its acetylation and deacetylation by the Gcn5p acetyltransferase and Rpd3p, respectively. Ume6p acetylation occurs in medium lacking dextrose and results in a partial destruction of the repressor. Preventing acetylation delays Ume6p meiotic destruction and retards both the transient transcription program and execution of the meiotic nuclear divisions. Conversely, mimicking acetylation induces partial destruction of Ume6p in dextrose medium and accelerates meiotic degradation by the APC/C. These studies reveal a new mechanism by which acetyltransferase activity induces gene expression through targeted destruction of a transcriptional repressor. These findings also demonstrate an important role for nonhistone acetylation in the transition between mitotic and meiotic cell division.
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http://dx.doi.org/10.1091/mbc.E11-06-0536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338428PMC
May 2012

Oxidative-stress-induced nuclear to cytoplasmic relocalization is required for Not4-dependent cyclin C destruction.

J Cell Sci 2012 Feb 15;125(Pt 4):1015-26. Epub 2012 Mar 15.

Department of Molecular Biology, University of Medicine and Dentistry New Jersey, Two Medical Center Drive, Stratford, NJ 08055, USA.

The yeast cyclin-C-Cdk8p kinase complex represses the transcription of a subset of genes involved in the stress response. To relieve this repression, cyclin C is destroyed in cells exposed to H(2)O(2) by the 26S proteasome. This report identifies Not4p as the ubiquitin ligase mediating H(2)O(2)-induced cyclin C destruction. Not4p is required for H(2)O(2)-induced cyclin C destruction in vivo and polyubiquitylates cyclin C in vitro by utilizing Lys48, a ubiquitin linkage associated with directing substrates to the 26S proteasome. Before its degradation, cyclin C, but not Cdk8p, translocates from the nucleus to the cytoplasm. This translocation requires both the cell-wall-integrity MAPK module and phospholipase C, and these signaling pathways are also required for cyclin C destruction. In addition, blocking cytoplasmic translocation slows the mRNA induction kinetics of two stress response genes repressed by cyclin C. Finally, a cyclin C derivative restricted to the cytoplasm is still subject to Not4p-dependent destruction, indicating that the degradation signal does not occur in the nucleus. These results identify a stress-induced proteolytic pathway regulating cyclin C that requires nuclear to cytoplasmic relocalization and Not4p-mediated ubiquitylation.
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http://dx.doi.org/10.1242/jcs.096479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311932PMC
February 2012

A disease-associated polymorphism alters splicing of the human CD45 phosphatase gene by disrupting combinatorial repression by heterogeneous nuclear ribonucleoproteins (hnRNPs).

J Biol Chem 2011 Jun 20;286(22):20043-53. Epub 2011 Apr 20.

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-8816, USA.

Alternative splicing is typically controlled by complexes of regulatory proteins that bind to sequences within or flanking variable exons. The identification of regulatory sequence motifs and the characterization of sequence motifs bound by splicing regulatory proteins have been essential to predicting splicing regulation. The activation-responsive sequence (ARS) motif has previously been identified in several exons that undergo changes in splicing upon T cell activation. hnRNP L binds to this ARS motif and regulates ARS-containing exons; however, hnRNP L does not function alone. Interestingly, the proteins that bind together with hnRNP L differ for different exons that contain the ARS core motif. Here we undertake a systematic mutational analysis of the best characterized context of the ARS motif, namely the ESS1 sequence from CD45 exon 4, to understand the determinants of binding specificity among the components of the ESS1 regulatory complex and the relationship between protein binding and function. We demonstrate that different mutations within the ARS motif affect specific aspects of regulatory function and disrupt the binding of distinct proteins. Most notably, we demonstrate that the C77G polymorphism, which correlates with autoimmune disease susceptibility in humans, disrupts exon silencing by preventing the redundant activity of hnRNPs K and E2 to compensate for the weakened function of hnRNP L. Therefore, these studies provide an important example of the functional relevance of combinatorial function in splicing regulation and suggest that additional polymorphisms may similarly disrupt function of the ESS1 silencer.
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http://dx.doi.org/10.1074/jbc.M111.218727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3103377PMC
June 2011

Signal- and development-dependent alternative splicing of LEF1 in T cells is controlled by CELF2.

Mol Cell Biol 2011 Jun 28;31(11):2184-95. Epub 2011 Mar 28.

Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 422 Curie Blvd, Philadelphia, PA 19104-6059, USA.

The HMG-box transcription factor LEF1 controls many developmentally regulated genes, including genes that activate expression of the T-cell antigen receptor alpha chain (TCR-alpha) in developing thymocytes. At least two distinct isoforms of LEF1 are expressed, resulting from variable inclusion of LEF1 exon 6; however, the expression pattern of these isoforms and mechanism of splicing regulation have not been explored. Here we demonstrate that inclusion of LEF1 exon 6 is increased during thymic development and in response to signaling in a cultured T-cell line in a manner which temporally correlates with increased expression of TCR-alpha. We further find that inclusion of exon 6 is dependent on the signal-induced increase in expression and binding of the splicing factor CELF2 to two intronic sequences flanking the regulated exon. Importantly, loss of exon 6 inclusion, through knockdown of CELF2 or direct block of the exon 6 splice site, results in reduced expression of TCR-alpha mRNA. Together, these data establish the mechanistic basis of LEF1 splicing regulation and demonstrate that LEF1 alternative splicing is a contributing determinant in the optimal expression of the TCR-alpha chain.
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http://dx.doi.org/10.1128/MCB.05170-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133246PMC
June 2011

Ume6p is required for germination and early colony development of yeast ascospores.

FEMS Yeast Res 2011 Feb 9;11(1):104-13. Epub 2010 Nov 9.

Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ 08055, USA.

Ume6p is a nonessential transcription factor that represses meiotic gene expression during vegetative growth in budding yeast. To relieve this repression, Ume6p is destroyed as cells enter meiosis and is not resynthesized until spore wall assembly. The present study reveals that spores derived from a ume6 null homozygous diploid fail to germinate. In addition, mutant spores from a UME6/ume6 heterozygote exhibited reduced germination efficiency compared with their wild-type sister spores. Analysis of ume6 spore colonies that did germinate revealed that the majority of cells in microcolonies following the first few cell divisions were inviable. As the colony developed, the viability percentage increased and achieved wild-type levels within approximately six cell divisions, indicating that the requirement for Ume6p in cell viability is transient. This function is specific for germinating spores as Ume6p has no or only a modest impact on the return to the growth ability of cells arrested at other points in the cell cycle. These results define a new role for Ume6p in spore germination and the first few subsequent mitotic cell divisions.
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http://dx.doi.org/10.1111/j.1567-1364.2010.00696.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951096PMC
February 2011

The Sin3p PAH domains provide separate functions repressing meiotic gene transcription in Saccharomyces cerevisiae.

Eukaryot Cell 2010 Dec 22;9(12):1835-44. Epub 2010 Oct 22.

Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Two Medical Center Drive, Stratford, NJ 08084, USA.

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.
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http://dx.doi.org/10.1128/EC.00143-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008282PMC
December 2010

Pds1p is required for meiotic recombination and prophase I progression in Saccharomyces cerevisiae.

Genetics 2009 Jan 10;181(1):65-79. Epub 2008 Nov 10.

Department of Biochemistry, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

Sister-chromatid separation at the metaphase-anaphase transition is regulated by a proteolytic cascade. Destruction of the securin Pds1p liberates the Esp1p separase, which ultimately targets the mitotic cohesin Mcd1p/Scc1p for destruction. Pds1p stabilization by the spindle or DNA damage checkpoints prevents sister-chromatid separation while mutants lacking PDS1 (pds1Delta) are temperature sensitive for growth due to elevated chromosome loss. This report examined the role of the budding yeast Pds1p in meiotic progression using genetic, cytological, and biochemical assays. Similar to its mitotic function, Pds1p destruction is required for metaphase I-anaphase I transition. However, even at the permissive temperature for growth, pds1Delta mutants arrest with prophase I spindle and nuclear characteristics. This arrest was partially suppressed by preventing recombination initiation or by inactivating a subset of recombination checkpoint components. Further studies revealed that Pds1p is required for recombination in both double-strand-break formation and synaptonemal complex assembly. Although deleting PDS1 did not affect the degradation of the meiotic cohesin Rec8p, Mcd1p was precociously destroyed as cells entered the meiotic program. This role is meiosis specific as Mcd1p destruction is not altered in vegetative pds1Delta cultures. These results define a previously undescribed role for Pds1p in cohesin maintenance, recombination, and meiotic progression.
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http://dx.doi.org/10.1534/genetics.108.095513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2621190PMC
January 2009

Meiosis-specific destruction of the Ume6p repressor by the Cdc20-directed APC/C.

Mol Cell 2007 Sep;27(6):951-61

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Meiotic development in yeast requires the coordinated induction of transient waves of gene transcription. The present study investigates the regulation of Ume6p, a mitotic repressor of the "early" class of meiosis-specific genes. Western blot analysis revealed that Ume6p is destroyed early in meiosis by Cdc20p, an activator of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. This control appears direct as Cdc20p and Ume6p associate in vivo and APC/C(Cdc20) ubiquitylates Ume6p in vitro. Inactivating Cdc20p, or stabilizing Ume6p through mutation, prevented meiotic gene transcription and meiotic progression. During mitotic cell division, Ume6p is protected from destruction by protein kinase A phosphorylation of Cdc20p. Complete elimination of Ume6p in meiotic cells requires association with the meiotic inducer Ime1p. These results indicate that Ume6p degradation is required for normal meiotic gene induction and meiotic progression. These findings demonstrate a direct connection between the transcription machinery and ubiquitin-mediated proteolysis that is developmentally regulated.
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http://dx.doi.org/10.1016/j.molcel.2007.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2034308PMC
September 2007

Regulation of the oxidative stress response through Slt2p-dependent destruction of cyclin C in Saccharomyces cerevisiae.

Genetics 2006 Mar 30;172(3):1477-86. Epub 2005 Dec 30.

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

The Saccharomyces cerevisiae C-type cyclin and its cyclin-dependent kinase (Cdk8p) repress the transcription of several stress response genes. To relieve this repression, cyclin C is destroyed in cells exposed to reactive oxygen species (ROS). This report describes the requirement of cyclin C destruction for the cellular response to ROS. Compared to wild type, deleting cyclin C makes cells more resistant to ROS while its stabilization reduces viability. The Slt2p MAP kinase cascade mediates cyclin C destruction in response to ROS treatment but not heat shock. This destruction pathway is important as deleting cyclin C suppresses the hypersensitivity of slt2 mutants to oxidative damage. The ROS hypersensitivity of an slt2 mutant correlates with elevated programmed cell death as determined by TUNEL assays. Consistent with the viability studies, the elevated TUNEL signal is reversed in cyclin C mutants. Finally, two results suggest that cyclin C regulates programmed cell death independently of its function as a transcriptional repressor. First, deleting its corepressor CDK8 does not suppress the slt2 hypersensitivity phenotype. Second, the human cyclin C, which does not repress transcription in yeast, does regulate ROS sensitivity. These findings demonstrate a new role for the Slt2p MAP kinase cascade in protecting the cell from programmed cell death through cyclin C destruction.
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http://dx.doi.org/10.1534/genetics.105.052266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1456298PMC
March 2006

Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast.

Genetics 2004 Aug;167(4):1621-8

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute.
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http://dx.doi.org/10.1534/genetics.104.029223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1470978PMC
August 2004

Ume1p represses meiotic gene transcription in Saccharomyces cerevisiae through interaction with the histone deacetylase Rpd3p.

J Biol Chem 2003 Nov 2;278(45):44727-34. Epub 2003 Sep 2.

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Ume1p is a member of a conserved protein family including RbAp48 that associates with histone deacetylases. Consistent with this finding, Ume1p is required for the full repression of a subset of meiotic genes during vegetative growth in budding yeast. In addition to mitotic cell division, this report describes a new role for Ume1p in meiotic gene repression in precommitment sporulating cultures returning to vegetative growth. However, Ume1p is not required to re-establish repression as part of the meiotic transient transcription program. Mutational analysis revealed that two conserved domains (NEE box and a WD repeat motif) are required for Ume1p-dependent repression. Co-immunoprecipitation studies revealed that both the NEE box and the WD repeat motif are essential for normal Rpd3p binding. Finally, Ume1p-Rpd3p association is dependent on the global co-repressor Sin3p. Moreover, this activity was localized to one of the four paired amphipathic-helix domains of Sin3p shown previously to be required for transcriptional repression. These findings support a model that Ume1p binding to Rpd3p is required for its repression activity. In addition, these results suggest that Rpd3-Ume1p-Sin3p comprises an interdependent complex required for mediating transcriptional repression.
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http://dx.doi.org/10.1074/jbc.M308632200DOI Listing
November 2003