scholarly journals Human TOB, an Antiproliferative Transcription Factor, Is a Poly(A)-Binding Protein-Dependent Positive Regulator of Cytoplasmic mRNA Deadenylation

2007 ◽  
Vol 27 (22) ◽  
pp. 7791-7801 ◽  
Author(s):  
Nader Ezzeddine ◽  
Tsung-Cheng Chang ◽  
Wenmiao Zhu ◽  
Akio Yamashita ◽  
Chyi-Ying A. Chen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Binding Protein ◽  
Processing Bodies ◽  
Protein Motifs ◽  
P Bodies ◽  
3T3 Fibroblasts ◽  
A Site

ABSTRACT In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3′ poly(A) tail-PABP complex.

scholarly journals CREB-Binding Protein Acetylates Hematopoietic Transcription Factor GATA-1 at Functionally Important Sites

1999 ◽  
Vol 19 (5) ◽  
pp. 3496-3505 ◽  
Author(s):  
Hsiao-Ling Hung ◽  
Jason Lau ◽  
Alexander Y. Kim ◽  
Mitchell J. Weiss ◽  
Gerd A. Blobel
Keyword(s):  
Transcription Factor ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Creb Binding Protein ◽  
Erythroid Cell Differentiation

ABSTRACT The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [3H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.

scholarly journals A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay

2005 ◽  
Vol 170 (6) ◽  
pp. 913-924 ◽  
Author(s):  
Maria A. Ferraiuolo ◽  
Sanjukta Basak ◽  
Josee Dostie ◽  
Elizabeth L. Murray ◽  
Daniel R. Schoenberg ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Binding Protein ◽  
Initiation Factor ◽  
Binding Motif ◽  
Processing Bodies ◽  
Mrna Decapping ◽  
P Bodies ◽  
Eukaryotic Initiation Factor 4E ◽  
Messenger Ribonucleoprotein ◽  
Bona Fide

4E-transporter (4E-T) is one of several proteins that bind the mRNA 5′cap-binding protein, eukaryotic initiation factor 4E (eIF4E), through a conserved binding motif. We previously showed that 4E-T is a nucleocytoplasmic shuttling protein, which mediates the import of eIF4E into the nucleus. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described processing bodies (P-bodies), which are believed to be sites of mRNA decay. In this paper, we demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies. Moreover, 4E-T controls mRNA half-life, because its depletion from cells using short interfering RNA increases mRNA stability. The 4E-T binding partner, eIF4E, also is localized in P-bodies. 4E-T interaction with eIF4E represses translation, which is believed to be a prerequisite for targeting of mRNAs to P-bodies. Collectively, these data suggest that 4E-T interaction with eIF4E is a priming event in inducing messenger ribonucleoprotein rearrangement and transition from translation to decay.

scholarly journals Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

scholarly journals The arrested state of processing bodies supports mRNA regulation in early development

2021 ◽  
Author(s):  
M. Sankaranarayanan ◽  
Ryan J. Emenecker ◽  
Marcus Jahnel ◽  
Irmela R. E. A. Trussina ◽  
Matt Wayland ◽  
...  
Keyword(s):  
Physical Properties ◽  
Electrostatic Interactions ◽  
Processing Bodies ◽  
P Bodies ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Dead Box ◽  
Premature Release ◽  
Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

On track with P-bodies

2010 ◽  
Vol 38 (1) ◽  
pp. 242-251 ◽  
Author(s):  
Meeta Kulkarni ◽  
Sevim Ozgur ◽  
Georg Stoecklin
Keyword(s):  
Light Microscopy ◽  
Mrna Decay ◽  
Molecular Level ◽  
Somatic Cells ◽  
Present Review ◽  
Dual Function ◽  
Processing Bodies ◽  
P Bodies ◽  
Specific Mrnas ◽  
Transient Interactions

P-bodies (processing bodies) are cytoplasmic foci visible by light microscopy in somatic cells of vertebrate and invertebrate origin as well as in yeast, plants and trypanosomes. At the molecular level, P-bodies are dynamic aggregates of specific mRNAs and proteins that serve a dual function: first, they harbour mRNAs that are translationally silenced, and such mRNA can exit again from P-bodies to re-engage in translation. Secondly, P-bodies recruit mRNAs that are targeted for deadenylation and degradation by the decapping/Xrn1 pathway. Whereas certain proteins are core constituents of P-bodies, others involved in recognizing short-lived mRNAs can only be trapped in P-bodies when mRNA decay is attenuated. This reflects the very transient interactions by which many proteins associate with P-bodies. In the present review, we summarize recent findings on the function, assembly and motility of P-bodies. An updated list of proteins and RNAs that localize to P-bodies will help in keeping track of this fast-growing field.

scholarly journals Regulation of Virulence of Entamoeba histolytica by the URE3-BP Transcription Factor

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Carol A. Gilchrist ◽  
Ellyn S. Moore ◽  
Yan Zhang ◽  
Christina B. Bousquet ◽  
Joanne A. Lannigan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Entamoeba Histolytica ◽  
Liver Abscess ◽  
Intestinal Epithelium ◽  
Binding Protein ◽  
Regulatory Element ◽  
Mutant Form ◽  
Content Type ◽  
Upstream Regulatory Element

ABSTRACTIt is not understood why only some infections withEntamoeba histolyticaresult in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison ofin vivotoin vitroparasite gene expression demonstrated that 39% ofin vivoregulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P< 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% ± 6% versus 14% ± 5%;P= 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P= 0.017), and a 3-fold increase in liver abscess size (0.1 ± 0.1 g versus 0.036 ± 0.1 g;P= 0.03). These results support a role for URE3-BP in virulence regulation.IMPORTANCEAmebic dysentery and liver abscess are caused byEntamoeba histolytica. Amebae colonize the colon and cause disease by invading the intestinal epithelium. However, only one in fiveE. histolyticainfections leads to disease. The factors that govern the transition from colonization to invasion are not understood. The transcription factor upstream regulatory element 3-binding protein (URE3-BP) is a calcium-responding regulator of theE. histolyticaGal/GalNAc lectin and ferredoxin genes, both implicated in virulence. Here we discovered that inducible expression of URE3-BP changed trophozoite morphology and promoted parasite invasion in the colon and liver. These results indicate that one determinant of virulence is transcriptional regulation by URE3-BP.

scholarly journals SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

2004 ◽  
Vol 24 (17) ◽  
pp. 7483-7490 ◽  
Author(s):  
Andrew Grimson ◽  
Sean O'Connor ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Null Alleles ◽  
Dependent Manner ◽  
Messenger Rnas ◽  
Phosphatidylinositol Kinase ◽  
Nonsense Mediated Mrna Decay

ABSTRACT Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

RNA granules and cytoskeletal links

2014 ◽  
Vol 42 (4) ◽  
pp. 1206-1210 ◽  
Author(s):  
Dipen Rajgor ◽  
Catherine M. Shanahan
Keyword(s):  
Mammalian Cells ◽  
Dynamic Properties ◽  
Translational Repression ◽  
Processing Bodies ◽  
P Bodies ◽  
Rna Granules ◽  
Messenger Ribonucleoprotein ◽  
Cytoskeletal Networks ◽  
And Control ◽  
Lower Eukaryote

In eukaryotic cells, non-translating mRNAs can accumulate into cytoplasmic mRNP (messenger ribonucleoprotein) granules such as P-bodies (processing bodies) and SGs (stress granules). P-bodies contain the mRNA decay and translational repression machineries and are ubiquitously expressed in mammalian cells and lower eukaryote species including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans. In contrast, SGs are only detected during cellular stress when translation is inhibited and form from aggregates of stalled pre-initiation complexes. SGs and P-bodies are related to NGs (neuronal granules), which are essential in the localization and control of mRNAs in neurons. Importantly, RNA granules are linked to the cytoskeleton, which plays an important role in mediating many of their dynamic properties. In the present review, we discuss how P-bodies, SGs and NGs are linked to cytoskeletal networks and the importance of these linkages in maintaining localization of their RNA cargoes.

scholarly journals Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon

1999 ◽  
Vol 19 (1) ◽  
pp. 78-85 ◽  
Author(s):  
Hua Lou ◽  
David M. Helfman ◽  
Robert F. Gagel ◽  
Susan M. Berget
Keyword(s):  
Binding Protein ◽  
Consensus Sequence ◽  
Polypyrimidine Tract ◽  
Terminal Exon ◽  
Exon Inclusion ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Exon 4 ◽  
A Site

ABSTRACT Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.

scholarly journals Poly(A) binding protein is required for mRNP remodeling to form P-bodies in mammalian cells

2021 ◽  
Author(s):  
Jingwei Xie ◽  
Yu Chen ◽  
Xiaoyu Wei ◽  
Guennadi Kozlov
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Stress Granules ◽  
Body Formation ◽  
P Bodies ◽  
Rna Granules ◽  
Cellular Processes ◽  
Early Stages ◽  
Summary Statement

AbstractCompartmentalization of mRNA through formation of RNA granules is involved in many cellular processes, yet it is not well understood. mRNP complexes undergo dramatic changes in protein compositions, reflected by markers of P-bodies and stress granules. Here, we show that PABPC1, albeit absent in P-bodies, plays important role in P-body formation. Depletion of PABPC1 decreases P-body population in unstressed cells. Upon stress in PABPC1 depleted cells, individual P-bodies fail to form and instead P-body proteins assemble on PABPC1-containing stress granules. We hypothesize that mRNP recruit proteins via PABPC1 to assemble P-bodies, before PABPC1 is displaced from mRNP. Further, we demonstrate that GW182 can mediate P-body assembly. These findings help us understand the early stages of mRNP remodeling and P-body formation.Summary statementA novel role of poly(A) binding protein is reported in P-body formation

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