scholarly journals Translation of human Δ133p53 mRNA and its targeting by antisense oligonucleotides complementary to the 5′-terminal region of this mRNA

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256938
Author(s):  
Paulina Żydowicz-Machtel ◽  
Mariola Dutkiewicz ◽  
Agata Swiatkowska ◽  
Dorota Gurda-Woźna ◽  
Jerzy Ciesiołka
Keyword(s):  
Secondary Structure ◽  
Antisense Oligonucleotides ◽  
Transcription Initiation ◽  
P53 Protein ◽  
Initiation Site ◽  
Terminal Region ◽  
Protein Isoforms ◽  
P53 Isoforms ◽  
P53 Mrna

The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5′-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5′-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5′ terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.

scholarly journals Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (11) ◽  
pp. e78863 ◽  
Author(s):  
Agnieszka Gorska ◽  
Agata Swiatkowska ◽  
Mariola Dutkiewicz ◽  
Jerzy Ciesiolka
Keyword(s):  
Antisense Oligonucleotides ◽  
Living Cells ◽  
Terminal Region ◽  
P53 Expression ◽  
P53 Mrna

Transcriptional and posttranscriptional regulation of maize mitochondrial gene expression

1991 ◽  
Vol 11 (1) ◽  
pp. 533-543
Author(s):  
R M Mulligan ◽  
P Leon ◽  
V Walbot
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Posttranscriptional Regulation ◽  
Rank Order ◽  
Mitochondrial Gene ◽  
Initiation Site ◽  
Gene Copy ◽  
Promoter Strength ◽  
Protein Coding

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Posterior stripe expression of hunchback is driven from two promoters by a common enhancer element

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 3067-3077 ◽  
Author(s):  
J.S. Margolis ◽  
M.L. Borowsky ◽  
E. Steingrimsson ◽  
C.W. Shim ◽  
J.A. Lengyel ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Embryo ◽  
Upstream Region ◽  
Enhancer Element ◽  
Gap Gene ◽  
Gap Genes ◽  
Two Phases ◽  
Necessary And Sufficient

The gap gene hunchback (hb) is required for the formation and segmentation of two regions of the Drosophila embryo, a broad anterior domain and a narrow posterior domain. Accumulation of hb transcript in the posterior of the embryo occurs in two phases, an initial cap covering the terminal 15% of the embryo followed by a stripe at the anterior edge of this region. By in situ hybridization with transcript-specific probes, we show that the cap is composed only of mRNA from the distal transcription initiation site (P1), while the later posterior stripe is composed of mRNA from both the distal and proximal (P2) transcription initiation sites. Using a series of genomic rescue constructs and promoter-lacZ fusion genes, we define a 1.4 kb fragment of the hb upstream region that is both necessary and sufficient for posterior expression. Sequences within this fragment mediate regulation by the terminal gap genes tailless (tll) and a huckebein, which direct the formation of the posterior hb stripe. We show that the tll protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by tll. We propose a model in which the anterior border of the posterior hb stripe is determined by tll concentration in a manner analogous to the activation of anterior hb expression by bicoid.

Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro

1985 ◽  
Vol 5 (2) ◽  
pp. 380-389
Author(s):  
S M Hanly ◽  
G C Bleecker ◽  
N Heintz
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
S Phase ◽  
Histone H4 ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Distal Promoter ◽  
Histone H4 Gene

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

scholarly journals Targeting cellular senescence in cancer and aging: roles of p53 and its isoforms

2020 ◽  
Vol 41 (8) ◽  
pp. 1017-1029 ◽  
Author(s):  
Jessica Beck ◽  
Casmir Turnquist ◽  
Izumi Horikawa ◽  
Curtis Harris
Keyword(s):  
Cellular Senescence ◽  
Therapeutic Potential ◽  
P53 Protein ◽  
Tp53 Gene ◽  
Suppressor Gene ◽  
Cellular Reprogramming ◽  
Protein Isoforms ◽  
Age Related ◽  
Alternative Mrna Splicing ◽  
P53 Isoforms

Abstract Cellular senescence and the associated secretory phenotype (SASP) promote disease in the aged population. Targeting senescent cells by means of removal, modulation of SASP or through cellular reprogramming represents a novel therapeutic avenue for treating cancer- and age-related diseases such as neurodegeneration, pulmonary fibrosis and renal disease. Cellular senescence is partly regulated by the TP53 gene, a critical tumor suppressor gene which encodes 12 or more p53 protein isoforms. This review marks a significant milestone of 40 years of Carcinogenesis publication history and p53 research and 15 years of p53 isoform research. The p53 isoforms are produced through initiation at alternative transcriptional and translational start sites and alternative mRNA splicing. These truncated p53 isoform proteins are endogenously expressed in normal human cells and maintain important functional roles, including modulation of full-length p53-mediated cellular senescence, apoptosis and DNA repair. In this review, we discuss the mechanisms and functions of cellular senescence and SASP in health and disease, the regulation of cellular senescence by p53 isoforms, and the therapeutic potential of targeting cellular senescence to treat cancer- and age-associated diseases.

scholarly journals Pathogenic bacterium Helicobacter pylori alters the expression profile of p53 protein isoforms and p53 response to cellular stresses

2012 ◽  
Vol 109 (38) ◽  
pp. E2543-E2550 ◽  
Author(s):  
Jinxiong Wei ◽  
Jennifer Noto ◽  
Elena Zaika ◽  
Judith Romero-Gallo ◽  
Pelayo Correa ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Expression Profile ◽  
P53 Protein ◽  
Pathogenic Bacterium ◽  
Protein Isoforms ◽  
Cellular Damage ◽  
Mongolian Gerbils ◽  
Cellular Stresses ◽  
H Pylori ◽  
P53 Isoforms

The p53 protein plays a central role in the prevention of tumorigenesis. Cellular stresses, such as DNA damage and aberrant oncogene activation, trigger induction of p53 that halts cellular proliferation and allows cells to be repaired. If cellular damage is beyond the capability of the repair mechanisms, p53 induces apoptosis or cell cycle arrest, preventing damaged cells from becoming cancerous. However, emerging evidence suggests that the function of p53 needs to be considered as isoform-specific. Here, we report that the expression profile of p53 can be shifted toward inhibitory p53 isoforms by the pathogenic bacterium Helicobacter pylori, which is known for its strong association with gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. We found that interaction of H. pylori with gastric epithelial cells, mediated via the cag pathogenicity island, induces N-terminally truncated ∆133p53 and ∆160p53 isoforms in human cells. Induction of an orthologous p53 isoform, ∆153p53, was also found in H. pylori-infected Mongolian gerbils. The p53 isoforms inhibit p53 and p73 activities, induce NF-κB, and increase survival of infected cells. Expression of ∆133p53, in response to H. pylori infection, is regulated by phosphorylation of c-Jun and activation of activator protein-1–dependent transcription. Together, these results provide unique insights into the regulation of p53 protein and may contribute to the understanding of tumorigenesis associated with H. pylori.

scholarly journals Loss of Pseudomonas aeruginosa PhpA Aminopeptidase Activity Results in Increased algDTranscription

2001 ◽  
Vol 183 (15) ◽  
pp. 4674-4679 ◽  
Author(s):  
Samuel C. Woolwine ◽  
April B. Sprinkle ◽  
Daniel J. Wozniak
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Dna Binding ◽  
Transcription Initiation ◽  
Leucine Aminopeptidase ◽  
Initiation Site ◽  
Aminopeptidase Activity ◽  
Homologous Protein ◽  
Relevant Property

ABSTRACT Inactivation of Pseudomonas aeruginosa phpA, encoding a putative leucine aminopeptidase, results in increased transcription ofalgD. The homologous protein in Escherichia coli, PepA, is multifunctional, possessing independent aminopeptidase and DNA-binding activities. Here we provide in vitro evidence that PhpA is an aminopeptidase and show that this activity is the relevant property with regard to algD expression. This regulation occurred at the previously mapped algDtranscription initiation site and was not due to activation of an alternative promoter.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

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