Posttranscriptional Regulation of Expression of the Gene for an Ammonium-Inducible Glutamate Dehydrogenase during the Cell Cycle of the Eukaryote Chlorella

AbstractBackgroundStaufen2 (STAU2) is an RNA binding protein involved in the posttranscriptional regulation of gene expression. In neurons, STAU2 is required to maintain the balance between differentiation and proliferation of neural stem cells through asymmetric cell division. However, the importance of controlling STAU2 expression for cell cycle progression is not clear in non-neuronal dividing cells. We recently showed that STAU2 transcription is inhibited in response to DNA-damage due to E2F1 displacement from theSTAU2gene promoter. We now study the regulation of STAU2 steady-state levels in unstressed cells and its consequence for cell proliferation.ResultsCRISPR/Cas9-mediated and RNAi-dependent STAU2 depletion in the non-transformed hTERT-RPE1 cells both facilitate cell proliferation suggesting that STAU2 expression influences pathway(s) linked to cell cycle controls. Such effects are not observed in the CRISPR STAU2-KO cancer HCT116 cells nor in the STAU2-RNAi-depleted HeLa cells. Interestingly, a physiological decrease in the steady-state level of STAU2 is controlled by caspases. This effect of peptidases is counterbalanced by the activity of the CHK1 pathway suggesting that STAU2 partial degradation/stabilization fines tune cell cycle progression in unstressed cells. A large-scale proteomic analysis using STAU2/biotinylase fusion protein identifies known STAU2 interactors involved in RNA translation, localization, splicing, or decay confirming the role of STAU2 in the posttranscriptional regulation of gene expression. In addition, several proteins found in the nucleolus, including proteins of the ribosome biogenesis pathway and of the DNA damage response, are found in close proximity to STAU2. Strikingly, many of these proteins are linked to the kinase CHK1 pathway, reinforcing the link between STAU2 functions and the CHK1 pathway. Indeed, inhibition of the CHK1 pathway for 4 h dissociates STAU2 from proteins involved in translation and RNA metabolism.ConclusionsThese results indicate that STAU2 is involved in pathway(s) that control(s) cell proliferation, likely via mechanisms of posttranscriptional regulation, ribonucleoprotein complex assembly, genome integrity and/or checkpoint controls. The mechanism by which STAU2 regulates cell growth likely involves caspases and the kinase CHK1 pathway.

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RAE-1 ligands for the NKG2D receptor are regulated by E2F transcription factors, which control cell cycle entry

Journal of Experimental Medicine ◽

10.1084/jem.20120565 ◽

2012 ◽

Vol 209 (13) ◽

pp. 2409-2422 ◽

Cited By ~ 77

Author(s):

Heiyoun Jung ◽

Benjamin Hsiung ◽

Kathleen Pestal ◽

Emily Procyk ◽

David H. Raulet

Keyword(s):

Cell Cycle ◽

Transcription Factors ◽

Stress Responses ◽

Transcriptional Activation ◽

Posttranscriptional Regulation ◽

Cellular Proliferation ◽

Control Cell ◽

T Cell Subsets ◽

Cell Cycle Entry

The NKG2D stimulatory receptor expressed by natural killer cells and T cell subsets recognizes cell surface ligands that are induced on transformed and infected cells and facilitate immune rejection of tumor cells. We demonstrate that expression of retinoic acid early inducible gene 1 (RAE-1) family NKG2D ligands in cancer cell lines and proliferating normal cells is coupled directly to cell cycle regulation. Raet1 genes are directly transcriptionally activated by E2F family transcription factors, which play a central role in regulating cell cycle entry. Induction of RAE-1 occurred in primary cell cultures, embryonic brain cells in vivo, and cells in healing skin wounds and, accordingly, wound healing was delayed in mice lacking NKG2D. Transcriptional activation by E2Fs is likely coordinated with posttranscriptional regulation by other stress responses. These findings suggest that cellular proliferation, as occurs in cancer cells but also other pathological conditions, is a key signal tied to immune reactions mediated by NKG2D-bearing lymphocytes.

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Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2350 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2350-2359 ◽

Cited By ~ 28

Author(s):

A Greco ◽

S S Gong ◽

M Ittmann ◽

C Basilico

Keyword(s):

Cell Cycle ◽

Housekeeping Genes ◽

Chloramphenicol Acetyltransferase ◽

Product Inhibition ◽

Asparagine Synthetase ◽

Cell Cycle Gene ◽

Mrna Levels ◽

Nonpermissive Temperature ◽

Regulation Of Expression ◽

Transcription Start Sites

The human ts11 gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle ts11 mutant, which is blocked in G1 at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the ts11 block. The ts11 (AS) mRNA has a size of about 2 kilobases and is induced in mid-G1 phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the ts11 gene. The human ts11 gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts11 P1) contained in a 280-base-pair region upstream of the first exon and the other (ts11 P2) contained in the first intron. ts11 P1 produced about sixfold more chloramphenicol acetyltransferase activity than did ts11 P2 and had features of the promoters of housekeeping genes: high G + C content, multiple transcription start sites, absence of a TATA box, and presence of putative Sp1 binding sites. ts11 P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The ts11 gene was overexpressed in ts11 cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented G1 induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.

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Control of Cell Cycle Progression in Human Natural Killer Cells Through Redox Regulation of Expression and Phosphorylation of Retinoblastoma Gene Product Protein

Blood ◽

10.1182/blood.v89.11.4092 ◽

1997 ◽

Vol 89 (11) ◽

pp. 4092-4099 ◽

Cited By ~ 31

Author(s):

Akira Yamauchi ◽

Eda T. Bloom

Keyword(s):

Cell Cycle ◽

Nk Cells ◽

Natural Killer ◽

Gene Product ◽

Redox Regulation ◽

Interleukin 2 ◽

Retinoblastoma Gene ◽

Regulation Of Expression ◽

Retinoblastoma Gene Product

Abstract Using thiol deprivation, we have previously shown that the response of natural killer (NK) cells to interleukin-2 (IL-2) is subject to redox regulation downstream of IL-2 binding and internalization. We have now used the IL-2–dependent cell line, NK3.3 to study redox regulation of NK cells further, and found that NK3.3 cells neither incorporated [3H]-thymidine nor completed the G1-S phase transition in medium lacking the thiol-related compounds, L-cystine, and glutathione, despite the presence of sufficient IL-2. Thiol deprivation did not alter the induction of DNA interferon-γ activated sequence (GAS)-binding activity in response to IL-2. However, the retinoblastoma gene product (RB), a cyclin-dependent kinase (CDK) substrate, was phosphorylated within 24 hours after IL-2 stimulation in standard medium, but its expression and phosphorylation were reduced in thiol-depleted medium in both NK3.3 cells and freshly isolated NK cells. These reductions were not associated with an increased level of p27Kip1, an inhibitor of CDKs CDK6/2 in association with G1 cyclins. Reducing agents, N-acetylcysteine, reduced glutathione or 2-ME restored both RB phosphorylation and DNA synthesis in thiol-deprived NK3.3 cells. The in vitro kinase activities of CDK6 and CDK2 were prematurely increased by thiol deprivation. This enhancement was associated with CDK hyperphosphorylation and prolonged phosphorylation, and could be observed before and beyond IL-2 stimulation. The data suggest the possibility that the premature and prolonged enhancement of CDK activity in thiol-deprived NK cells is associated with, and therefore may contribute to, the reduced expression and phosphorylation of RB, and the associated cell cycle arrest.

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Role of transcriptional and posttranscriptional regulation in expression of histone genes in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.614-621.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 614-621

Author(s):

D E Lycan ◽

M A Osley ◽

L M Hereford

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Posttranscriptional Regulation ◽

Cell Cycle Control ◽

Dimensional Structure ◽

Histone Genes ◽

Transcriptional Regulatory Mechanisms ◽

Rna Levels

We analyzed the role of posttranscriptional mechanisms in the regulation of histone gene expression in Saccharomyces cerevisiae. The rapid drop in histone RNA levels associated with the inhibition of ongoing DNA replication was postulated to be due to posttranscriptional degradation of histone transcripts. However, in analyzing the sequences required for this response, we showed that the coupling of histone RNA levels to DNA replication was due mostly, if not entirely, to transcriptional regulatory mechanisms. Furthermore, deletions which removed the negative, cell cycle control sequences from the histone promoter also uncoupled histone transcription from DNA replication. We propose that the arrest of DNA synthesis prematurely activates the regulatory pathway used in the normal cell cycle to repress transcription. Although posttranscriptional regulation did not appear to play a significant role in coupling histone RNA levels to DNA replication, it did affect the levels of histone RNA in the cell cycle. Posttranscriptional regulation could apparently restore much of the periodicity of histone RNA accumulation in cells which constitutively transcribed the histone genes. Unlike transcriptional regulation, periodic posttranscriptional regulation appears to operate on a clock which is independent of events in the mitotic DNA cycle. Posttranscriptional recognition of histone RNA must require either sequences in the 3' end of the RNA or an intact three-dimensional structure since H2A- and H2B-lacZ fusion transcripts, containing only 5' histone sequences, were insensitive to posttranscriptional controls.

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Cell cycle- and terminal differentiation-associated regulation of the mouse mRNA encoding a conserved mitotic protein kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7793-7801.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7793-7801

Author(s):

R J Lake ◽

W R Jelinek

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Posttranscriptional Regulation ◽

Terminal Differentiation ◽

The Body ◽

Erythroleukemia Cells ◽

M Phase ◽

Erythroleukemia Cell ◽

Low Levels ◽

Murine Erythroleukemia

We determined the nucleotide sequence of a mouse and a human cDNA, which we designate STPK13, that encodes an apparent protein kinase related to that encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene. The polo and CDC5 gene products are required for normal mitosis. The STPK13 mRNA is regulated during terminal erythrodifferentiation and during the cell cycle. Within the precommitment period of murine erythroleukemia cell terminal differentiation, most of the poly(A) tail is lost from the STPK13 mRNA, but the body of the mRNA remains unchanged in abundance; this poly(A) loss does not occur in mutant erythroleukemia cells that fail to commit to terminal differentiation. During the cell cycle, the abundance of the body of the STPK13 mRNA fluctuates. The mRNA is present in growing but not in nongrowing cells. It reaches a maximum abundance during G2/M phase, is absent or present at only low levels during G1 phase, and begins to reaccumulate at approximately the middle of S phase. The cell cycle-associated accumulation and loss of the STPK13 mRNA could cause a similar fluctuation in abundance of its encoded protein kinase, thereby providing a maximum amount during M phase, when the kinase is thought to function, and little or none at other times of the cell cycle. Posttranscriptional regulation must be responsible for the cell cycle-associated fluctuations because transcription rates are relatively constant during different times of the cell cycle when there are large differences in mRNA abundance.

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Phosphorylation of Stem-Loop Binding Protein (SLBP) on Two Threonines Triggers Degradation of SLBP, the Sole Cell Cycle-Regulated Factor Required for Regulation of Histone mRNA Processing, at the End of S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.23.5.1590-1601.2003 ◽

2003 ◽

Vol 23 (5) ◽

pp. 1590-1601 ◽

Cited By ~ 62

Author(s):

Lianxing Zheng ◽

Zbigniew Dominski ◽

Xiao-Cui Yang ◽

Phillip Elms ◽

Christy S. Raska ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Posttranscriptional Regulation ◽

Binding Protein ◽

S Phase ◽

Mrna Processing ◽

Histone Mrna ◽

Stem Loop ◽

Histone Mrnas ◽

Nuclear Extracts

ABSTRACT The replication-dependent histone mRNAs, the only eukaryotic mRNAs that do not have poly(A) tails, are present only in S-phase cells. Coordinate posttranscriptional regulation of histone mRNAs is mediated by the stem-loop at the 3′ end of histone mRNAs. The protein that binds the 3′ end of histone mRNA, stem-loop binding protein (SLBP), is required for histone pre-mRNA processing and is involved in multiple aspects of histone mRNA metabolism. SLBP is also regulated during the cell cycle, accumulating as cells enter S phase and being rapidly degraded as cells exit S phase. Mutation of any residues in a TTP sequence (amino acids 60 to 62) or mutation of a consensus cyclin binding site (amino acids 99 to 104) stabilizes SLBP in G2 and mitosis. These two threonines are phosphorylated in late S phase, as determined by mass spectrometry (MS) of purified SLBP from late S-phase cells, triggering SLBP degradation. Cells that express a stable SLBP still degrade histone mRNA at the end of S phase, demonstrating that degradation of SLBP is not required for histone mRNA degradation. Nuclear extracts from G1 and G2 cells are deficient in histone pre-mRNA processing, which is restored by addition of recombinant SLBP, indicating that SLBP is the only cell cycle-regulated factor required for histone pre-mRNA processing.

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A common cis-element in promoters of protein synthesis and cell cycle genes.

Acta Biochimica Polonica ◽

10.18388/abp.2007_3273 ◽

2007 ◽

Vol 54 (1) ◽

pp. 89-98 ◽

Cited By ~ 13

Author(s):

Lucjan S Wyrwicz ◽

Paweł Gaj ◽

Marcin Hoffmann ◽

Leszek Rychlewski ◽

Jerzy Ostrowski

Keyword(s):

Cell Cycle ◽

Protein Synthesis ◽

Regulatory Element ◽

Experimental Methodology ◽

Gene Promoters ◽

Ribosomal Protein Genes ◽

Regulation Of Expression ◽

Cell Cycle Genes ◽

Essential Components ◽

Sequence Elements

Gene promoters contain several classes of functional sequence elements (cis elements) recognized by protein agents, e.g. transcription factors and essential components of the transcription machinery. Here we describe a common DNA regulatory element (tandem TCTCGCGAGA motif) of human TATA-less promoters. A combination of bioinformatic and experimental methodology suggests that the element can be critical for expression of genes involved in enhanced protein synthesis and the G1/S transition in the cell cycle. The motif was identified in a substantial fraction of promoters of cell cycle genes, like cyclins (CCNC, CCNG1), as well as transcription regulators (TAF7, TAF13, KLF7, NCOA2), chromatin structure modulators (HDAC2, TAF6L), translation initiation factors (EIF5, EIF2S1, EIF4G2, EIF3S8, EIF4) and previously reported 18 ribosomal protein genes. Since the motif can define a subset of promoters with a distinct mechanism of activation involved in regulation of expression of about 5% of human genes, further investigation of this regulatory element is an emerging task.

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MicroRNA regulation of murine trophoblast stem cell self-renewal and differentiation

Life Science Alliance ◽

10.26508/lsa.202000674 ◽

2020 ◽

Vol 3 (11) ◽

pp. e202000674

Author(s):

Sarbani Saha ◽

Rupasri Ain

Keyword(s):

Cell Cycle ◽

Cyclin D1 ◽

Posttranscriptional Regulation ◽

Es Cells ◽

Placental Development ◽

Self Renewal ◽

Microrna Regulation ◽

Cyclin E1 ◽

Trophoblast Stem ◽

Mirna Clusters

Proper placentation is fundamental to successful pregnancy. Placenta arises from differentiation of trophoblast stem (TS) cells during development. Despite being recognized as the counterpart of ES cells in placental development, the role of regulatory miRNAs in TS cell differentiation remains inadequately explored. Here, we have identified complete repertoire of microRNAs present in mouse trophoblast cells in proliferative and differentiated state. We demonstrated that two miRNA clusters, -290 and -322, displayed reciprocal expression during trophoblast differentiation. Loss of miR-290 cluster members or gain in miR-322 cluster members led to differentiation of TS cells. The trophoblast stemness factor, CDX2, transactivated the miR-290 cluster and Cyclin D1. MiR-290 cluster members repressed cell cycle repressors, P21, P27, WEE1, RBL2, and E2F7, in TS cells. MiR-322 cluster members repressed the cell cycle activators, CYCLIN D1, CYCLIN E1, CDC25B, and CDX2, to induce differentiation. Taken together, our findings highlight the importance of posttranscriptional regulation by conserved miRNA clusters that form a regulatory network with CDX2, cell cycle activators, and repressors in equipoising TS cell self-renewal and differentiation.

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