scholarly journals The Herpesvirus saimiri Small Nuclear RNAs Recruit AU-Rich Element-Binding Proteins but Do Not Alter Host AU-Rich Element-Containing mRNA Levels in Virally Transformed T Cells

2004 ◽  
Vol 24 (10) ◽  
pp. 4522-4533 ◽  
Author(s):  
Heidi L. Cook ◽  
Hannah E. Mischo ◽  
Joan A. Steitz
Keyword(s):  
T Cells ◽  
Posttranscriptional Regulation ◽  
Binding Proteins ◽  
Herpesvirus Saimiri ◽  
Mrna Levels ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Latently Infected ◽  
The Stability

ABSTRACT Herpesvirus saimiri (HVS) encodes seven Sm-class small nuclear RNAs, called HSURs (for Herpesvirus saimiri U RNAs), that are abundantly expressed in HVS-transformed, latently infected marmoset T cells but are of unknown function. HSURs 1, 2, and 5 have highly conserved 5′-end sequences containing the AUUUA pentamer characteristic of AU-rich elements (AREs) that regulate the stability of many host mRNAs, including those encoding most proto-oncogenes and cytokines. To test whether the ARE-containing HSURs act to sequester host proteins that regulate the decay of these mRNAs, we demonstrate their in vivo interaction with the ARE-binding proteins hnRNP D and HuR in HVS-transformed T cells using a new cross-linking assay. Comprehensive Northern and microarray analyses revealed, however, that the levels of endogenous ARE-containing mRNAs are not altered in T cells latently infected with HVS mutants lacking HSURs 1 and 2. HSUR 1 binds the destabilizing ARE-binding protein tristetraprolin induced following activation of HVS-transformed T cells, but even in such stimulated cells, the levels of host ARE-containing mRNAs are not altered by deletion of HSURs 1 and 2. Instead, HSUR 1 itself is degraded by an ARE-dependent pathway in HVS-transformed T cells, suggesting that HVS may take advantage of the host ARE-mediated mRNA decay pathway to regulate HSUR expression. This is the first example of posttranscriptional regulation of the expression of an Sm small nuclear RNA.

scholarly journals Transcriptional and posttranscriptional regulation of the interleukin-4 and interleukin-3 genes in human T cells

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 35-40 ◽  
Author(s):  
WH Dokter ◽  
MT Esselink ◽  
SJ Sierdsema ◽  
MR Halie ◽  
E Vellenga
Keyword(s):  
T Cells ◽  
Mrna Expression ◽  
Posttranscriptional Regulation ◽  
Interleukin 4 ◽  
Mrna Levels ◽  
Con A ◽  
Mrna Accumulation ◽  
Human T Cells ◽  
Activation Pathways ◽  
The Stability

Abstract Human T cells were studied with regard to the regulation of interleukin- 4 (IL-4) and IL-3 gene expression. IL-4 and IL-3 mRNA were undetectable in unstimulated T cells. On activation with the lectin concanavalin A (Con A), both IL-4 and IL-3 mRNA were expressed. Accumulation of IL-4 mRNA peaked after 6 to 12 hours, whereas IL-3 mRNA levels peaked after 3 to 6 hours of stimulation with Con A. Nuclear run-on assays showed a low constitutive transcription for both genes. The transcription rates were increased by Con A resulting in a peak for IL-4 after 1 hour (30% increase) and for IL-3 after 3 hours (40% increase) of Con A treatment. mRNA stability studies demonstrated that on activation with Con A both messages decayed with a half-life of approximately 90 minutes. No IL-4 or IL-3 mRNA expression was induced by the protein kinase C activator phorbol myristate acetate (PMA). However, PMA augmented the Con A- induced IL-4 and IL-3 mRNA accumulation. This was shown to be mediated at posttranscriptional level by a large increase in the stability of both messages (t 1/2 > 3 hours). The transcription rate of both genes was also enhanced by Con A+PMA and reached peak levels for IL-4 after 1 hour (90% increase) and for IL-3 after 3 hours (70% increase) of stimulation. Furthermore, it appeared that the induction of IL-4 mRNA was dependent on protein synthesis because cycloheximide (CHX) blocked the Con A- and Con A+PMA-induced expression of IL-4 mRNA. In contrast, CHX inhibited, but failed to completely block, the Con A- and Con A+PMA- induced IL-3 mRNA expression, whereas the expression of both genes was completely blocked by cyclosporine A. With regard to the secretion of IL-4 protein it was shown that it closely follows the accumulation of IL-4 mRNA. Taken together, the data show that expression of the IL-4 and IL-3 genes in human T cells is controlled by different activation pathways that affect the gene regulation at transcriptional and posttranscriptional levels.

scholarly journals Transcriptional and posttranscriptional regulation of the interleukin-4 and interleukin-3 genes in human T cells

Blood ◽  
1993 ◽  
Vol 81 (1) ◽  
pp. 35-40
Author(s):  
WH Dokter ◽  
MT Esselink ◽  
SJ Sierdsema ◽  
MR Halie ◽  
E Vellenga
Keyword(s):  
T Cells ◽  
Mrna Expression ◽  
Posttranscriptional Regulation ◽  
Interleukin 4 ◽  
Mrna Levels ◽  
Con A ◽  
Mrna Accumulation ◽  
Human T Cells ◽  
Activation Pathways ◽  
The Stability

Human T cells were studied with regard to the regulation of interleukin- 4 (IL-4) and IL-3 gene expression. IL-4 and IL-3 mRNA were undetectable in unstimulated T cells. On activation with the lectin concanavalin A (Con A), both IL-4 and IL-3 mRNA were expressed. Accumulation of IL-4 mRNA peaked after 6 to 12 hours, whereas IL-3 mRNA levels peaked after 3 to 6 hours of stimulation with Con A. Nuclear run-on assays showed a low constitutive transcription for both genes. The transcription rates were increased by Con A resulting in a peak for IL-4 after 1 hour (30% increase) and for IL-3 after 3 hours (40% increase) of Con A treatment. mRNA stability studies demonstrated that on activation with Con A both messages decayed with a half-life of approximately 90 minutes. No IL-4 or IL-3 mRNA expression was induced by the protein kinase C activator phorbol myristate acetate (PMA). However, PMA augmented the Con A- induced IL-4 and IL-3 mRNA accumulation. This was shown to be mediated at posttranscriptional level by a large increase in the stability of both messages (t 1/2 > 3 hours). The transcription rate of both genes was also enhanced by Con A+PMA and reached peak levels for IL-4 after 1 hour (90% increase) and for IL-3 after 3 hours (70% increase) of stimulation. Furthermore, it appeared that the induction of IL-4 mRNA was dependent on protein synthesis because cycloheximide (CHX) blocked the Con A- and Con A+PMA-induced expression of IL-4 mRNA. In contrast, CHX inhibited, but failed to completely block, the Con A- and Con A+PMA- induced IL-3 mRNA expression, whereas the expression of both genes was completely blocked by cyclosporine A. With regard to the secretion of IL-4 protein it was shown that it closely follows the accumulation of IL-4 mRNA. Taken together, the data show that expression of the IL-4 and IL-3 genes in human T cells is controlled by different activation pathways that affect the gene regulation at transcriptional and posttranscriptional levels.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

scholarly journals Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

1987 ◽  
Vol 7 (12) ◽  
pp. 4357-4368 ◽  
Author(s):  
N G Theodorakis ◽  
R I Morimoto
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Posttranscriptional Regulation ◽  
High Efficiency ◽  
Hsp70 Expression ◽  
Mrna Levels ◽  
Protein Synthesis Inhibitors ◽  
Hsp70 Mrna ◽  
Infected Cells ◽  
The Stability

We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated.

Toll-like receptor–induced reactivity and strongly potentiated IL-8 production in granulocytes mobilized for transfusion purposes

Blood ◽  
2010 ◽  
Vol 115 (22) ◽  
pp. 4588-4596 ◽  
Author(s):  
Agata Drewniak ◽  
Anton T. J. Tool ◽  
Judy Geissler ◽  
Robin van Bruggen ◽  
Timo K. van den Berg ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Cytokine Production ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Mrna Levels ◽  
Granulocyte Colony Stimulating Factor ◽  
Toll Like Receptor ◽  
Cellular Behavior ◽  
Gene And Protein Expression ◽  
Stimulating Factor

AbstractTransfusion of granulocytes from granulocyte-colony stimulating factor (G-CSF)/dexamethasone (dexa)–treated donors can be beneficial for neutropenic recipients that are refractory to antimicrobial therapy. G-CSF/dexa treatment not only increases the number of circulating neutrophils but also affects their gene expression. Because of the intended transfusion of these granulocytes into patients who are severely ill, it is of importance to establish to what extent mobilization affects the cellular behavior of neutrophils. Here, we studied the effects of mobilization on Toll-like receptor (TLR)–mediated responses. Mobilized granulocytes displayed increased gene and protein expression of TLR2, TLR4, TLR5, and TLR8. Although mobilized granulocytes displayed normal priming of nicotinamide adenine dinucleotide phosphate oxidase activity and a slight increase in adhesion in response to TLR stimulation, these cells produced massive amounts of interleukin-8 (IL-8), in particular to TLR2 and TLR8 stimulation. The increase in IL-8 release occurred despite reduced IL-8 mRNA levels in the donor granulocytes after in vivo G-CSF/dexa treatment, indicating that the enhanced TLR-induced IL-8 production was largely determined by posttranscriptional regulation. In summary, granulocytes mobilized for transfusion purposes show enhanced TLR responsiveness in cytokine production, which is anticipated to be beneficial for the function of these cells on transfusion into patients.

Splicing signals and factors in plant intron removal

2002 ◽  
Vol 30 (2) ◽  
pp. 146-149 ◽  
Author(s):  
J. W. S. Brown ◽  
C. G. Simpson ◽  
G. Thow ◽  
G. P. Clark ◽  
S. N. Jennings ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Viral Vectors ◽  
Binding Proteins ◽  
Fluorescent Protein ◽  
Splicing Factors ◽  
Small Nuclear Rna ◽  
Intron Removal ◽  
Arabidopsis Proteins ◽  
Green Fluorescent

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

scholarly journals RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins

1994 ◽  
Vol 14 (2) ◽  
pp. 1230-1244
Author(s):  
W Reith ◽  
C Ucla ◽  
E Barras ◽  
A Gaud ◽  
B Durand ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Dna Binding ◽  
Hepatitis B ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Mrna Levels ◽  
B Virus ◽  
Factor C

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

scholarly journals Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals

2020 ◽  
Vol 16 (11) ◽  
pp. e1009060
Author(s):  
Sara Moron-Lopez ◽  
Sushama Telwatte ◽  
Indra Sarabia ◽  
Emilie Battivelli ◽  
Mauricio Montano ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Primary Cell ◽  
Cell Models ◽  
Transcriptional Initiation ◽  
Cellular Genes ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv

It is unclear what mechanisms govern latent HIV infection in vivo or in primary cell models. To investigate these questions, we compared the HIV and cellular transcription profile in three primary cell models and peripheral CD4+ T cells from HIV-infected ART-suppressed individuals using RT-ddPCR and RNA-seq. All primary cell models recapitulated the block to HIV multiple splicing seen in cells from ART-suppressed individuals, suggesting that this may be a key feature of HIV latency in primary CD4+ T cells. Blocks to HIV transcriptional initiation and elongation were observed more variably among models. A common set of 234 cellular genes, including members of the minor spliceosome pathway, was differentially expressed between unstimulated and activated cells from primary cell models and ART-suppressed individuals, suggesting these genes may play a role in the blocks to HIV transcription and splicing underlying latent infection. These genes may represent new targets for therapies designed to reactivate or silence latently-infected cells.

scholarly journals Antigenic Restimulation of Virus-Specific Memory CD8+ T Cells Requires Days of Lytic Protein Accumulation for Maximal Cytotoxic Capacity

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Stephen A. Migueles ◽  
Daniel C. Rogan ◽  
Noah V. Gavil ◽  
Elizabeth P. Kelly ◽  
Sushila A. Toulmin ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Expression ◽  
Posttranscriptional Regulation ◽  
Granzyme B ◽  
Protein Accumulation ◽  
Mrna Levels ◽  
Memory Cells ◽  
Virus Infections ◽  
Prolonged Stimulation ◽  
Specific Memory

ABSTRACT In various infections or vaccinations of mice or humans, reports of the persistence and the requirements for restimulation of the cytotoxic mediators granzyme B (GrB) and perforin (PRF) in CD8+ T cells have yielded disparate results. In this study, we examined the kinetics of PRF and GrB mRNA and protein expression after stimulation and associated changes in cytotoxic capacity in virus-specific memory cells in detail. In patients with controlled HIV or cleared respiratory syncytial virus (RSV) or influenza virus infections, all virus-specific CD8+ T cells expressed low PRF levels without restimulation. Following stimulation, they displayed similarly delayed kinetics for lytic protein expression, with significant increases occurring by days 1 to 3 before peaking on days 4 to 6. These increases were strongly correlated with, but were not dependent upon, proliferation. Incremental changes in PRF and GrB percent expression and mean fluorescence intensity (MFI) were highly correlated with increases in HIV-specific cytotoxicity. mRNA levels in HIV-specific CD8+ T-cells exhibited delayed kinetics after stimulation as with protein expression, peaking on day 5. In contrast to GrB, PRF mRNA transcripts were little changed over 5 days of stimulation (94-fold versus 2.8-fold, respectively), consistent with posttranscriptional regulation. Changes in expression of some microRNAs, including miR-17, miR-150, and miR-155, suggested that microRNAs might play a significant role in regulation of PRF expression. Therefore, under conditions of extremely low or absent antigen levels, memory virus-specific CD8+ T cells require prolonged stimulation over days to achieve maximal lytic protein expression and cytotoxic capacity. IMPORTANCE Antigen-specific CD8+ T cells play a major role in controlling most virus infections, primarily by perforin (PRF)- and granzyme B (GrB)-mediated apoptosis. There is considerable controversy regarding whether PRF is constitutively expressed, rapidly increased similarly to a cytokine, or delayed in its expression with more prolonged stimulation in virus-specific memory CD8+ T cells. In this study, the degree of cytotoxic capacity of virus-specific memory CD8+ T cells was directly proportional to the content of lytic molecules, which required antigenic stimulation over several days for maximal levels. This appeared to be modulated by increases in GrB transcription and microRNA-mediated posttranscriptional regulation of PRF expression. Clarifying the requirements for maximal cytotoxic capacity is critical to understanding how viral clearance might be mediated by memory cells and what functions should be induced by vaccines and immunotherapies.

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