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Results for “RNA Stability”

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At least 811 records · Page 45Linked to original sources

PubMed2006

Characterization of salivary RNA by cDNA library analysis.

Oral fluid (saliva) meets the demands for a noninvasive and accessible diagnostic medium. Recent reports by our group and others described the presence and use of human RNA in saliva as a diagnostic or forensic tool, including the use for oral cancer detection. To gain insights into the integrity of salivary RNA, we examined in detail the integrity of salivary RNA by generating a cDNA library from pooled supernatant saliva of 10 healthy donors. From a library with a primary library titer of 1.3 x 10(6) cfu/mL of which 95% of the clones had inserts, we successfully sequenced 117 random colonies containing recombinant clones. BLAST search results indicated that all of these clones contained sequences of human origin. Most of the salivary RNAs appeared to be endonucleolytically cleaved at random positions as indicated by comparisons to respective full length parental RNAs from the Genbank. Twelve of the insert sequences matched to the normal salivary core transcriptome sequences, which are highly abundant mRNAs present in healthy individuals. This study provides an in-depth molecular analysis of the saliva transcriptome and should be a useful resource for future basic and translational studies of RNA in human saliva. In addition, this paper presents unequivocal evidence for the presence of RNA in saliva as determined by the use of diverse techniques such as reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in vitro translation, and the construction of a salivary cDNA library.

Adult↗

PubMed2006

RhoA-dependent PAI-1 gene expression induced in endothelial cells by monocyte adhesion mediates geranylgeranyl transferase I and Ca2+ signaling.

We investigated the role of RhoA activation and its mechanism in plasminogen activator inhibitor-1 (PAI-1) gene expression induced in endothelial cells by monocyte adhesion. Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased PAI-1 expression at the transcriptional level and activated RhoA which was accompanied by an increase in the activity of geranylgeranyl transferase I (GGTase I), an enzyme responsible for geranylgeranylation, and actin stress fiber formation. Inhibition of RhoA by C3 exoenzyme or by adenovirus-mediated expression of N19RhoA, as well as by pravastatin, prevented the upregulation of PAI-1 induced by monocyte adhesion. GGTI-286, an inhibitor of GGTase I, prevented the monocyte-induced RhoA activation and PAI-1 expression in endothelial cells. Monocyte attachment induced an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells and Ca(2+) chelation prevented the increased promoter activity and expression of PAI-1 induced by monocyte adhesion. C3 exoenzyme and GGTI-286 also suppressed endothelial intracellular Ca(2+) mobilization and Ca(2+) entry induced by monocytes. The present study shows that GGTase I plays a role in the RhoA activation in endothelial cells induced by monocyte adhesion and that GGTase I-mediated Ca(2+) signaling may contribute to RhoA-dependent PAI-1 gene expression.

Actins↗

PubMed2006

Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms.

The present study aimed to characterize mutant alleles in Mucopolysaccharidosis II and evaluate possible reduction of mRNA amount consequent to nonsense-mediated or nonstop mRNA decay pathways. A combination of different approaches, including real-time RT-PCR, were used to molecularly characterize seventeen patients. Fifteen alleles were identified and nine of them were new. The novel alleles consisted of three missense mutations (p.S71R, p.P197R, p.C432R), two nonsense (p.Q66X, p.L359X), two frameshifts (p.V136fs75X, p.C432fs8X), one allele carrying two in-cis mutations [p.D252N;p.S369X], and a large deletion (p.G394_X551). Analysing these results it emerged that most of the alterations resulted in mutants leading to mRNAs with premature termination codons, and therefore, potentially sensitive to mRNA surveillance pathway. By using real-time RT-PCR, the mRNAs resulting (i) from substitutions that changed one amino acid to a stop codon (L359X, and S369X), or caused the shifted reading frame with premature introduction of a stop codon (C432fs8X), (ii) from large deletion (p.G394_X551) that included the termination codon, seemed to be subject to degradation by nonsense-mediated (i) or nonstop decay (ii) mechanisms, as mRNA was strongly underexpressed. On the contrary, two mutations (Q66X and V136fs75X) produced transcripts evading mRNA surveillance pathway despite both of them fulfilled the known criteria. These results confirm the wide variability of the mRNA expression levels previously reported and represent a further exception to the rules governing susceptibility to nonsense-mediated decay. A close examination of the molecular basis of the disease is becoming increasingly important for optimising the choices of available or forthcoming therapies such as, enzyme replacement therapy or enzyme enhancement therapy.

Alleles↗

PubMed2003

tRNA hopping: effects of mutant tRNAs.

Movement of tRNA and mRNA through the ribosome is coupled. However, selection for suppression of a -1 frameshift mutation in Escherichia coli has yielded a class of mutant tRNAs that can violate this mechanism and "hop" or disengage from their cognate codons and re-pair downstream in the mRNA. Previously described tRNA mutants of this class included those with insertions in the anticodon of tRNA(Val)1. This report describes further tRNA(Val)1 alterations that enhance hopping; these include a novel insertion in the anticodon loop, base substitutions in the anticodon stem and a base deletion in the variable loop. These results indicate that several different features of a tRNA are important for maintaining stable codon-anticodon interactions and coupled movement of tRNA and mRNA during the elongation phase of protein synthesis.

Anticodon↗

PubMed2005

The role of SMG-1 in nonsense-mediated mRNA decay.

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.

Animals↗

PubMed2004

Guideline to reference gene selection for quantitative real-time PCR.

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and beta-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.

Base Sequence↗

PubMed2004

Inhibition of the functions of the nucleocapsid protein of human immunodeficiency virus-1 by an RNA aptamer.

The nucleocapsid (NC) protein plays many roles in the life cycle of human immunodeficiency virus type-1 (HIV-1). Previously we selected the NC binding RNA aptamers from diverse forms of RNA libraries. Here we used one of the RNA aptamers to the NC protein, N70-13, and tested its effect on NC protein in vitro and in cells. The high affinity RNA aptamer completely abolished NC binding to the stable transactivation response hairpin and psi RNA stem-loops of HIV-1 RNA. When it was expressed in cells as an intramer it inhibited the packaging of viral genomic RNA and therefore promises to be an effective anti-HIV therapeutic tool.

Binding Sites↗

PubMed2004

Cytoplasmic nonsense-mediated mRNA decay for a nonsense (W262X) transcript of the gene responsible for hereditary tyrosinemia, fumarylacetoacetate hydrolase.

Messenger RNAs containing premature stop codons are generally targeted for degradation through the nonsense-mediated mRNA decay (NMD) pathway. The subcellular localization of the NMD process in higher eukaryotes remains controversial. While many mRNAs are subjected to NMD prior to their release from the nucleus, a few display cytoplasmic NMD. To understand the possible impact of NMD on the pathogenesis of hereditary tyrosinemia type I, a severe metabolic disease caused by fumarylacetoacetate hydrolase (FAH) deficiency, we examined the metabolism of FAH mRNA harboring a nonsense mutation, W262X, in lymphoblastoid cell lines derived from patients and their parents. W262X-FAH transcripts show a approximately 20-fold reduction in abundance in mutant cells, which is translation-dependent. Cellular fractionation shows that this down-regulation of the W262X transcript occurs in the cytoplasm. Thus, the W262X FAH is another example of nonsense mRNAs subjected to the NMD pathway in the cytoplasm.

Cell Line↗

PubMed2005

E2F1 up-regulates the expression of the tumour suppressor axin2 both by activation of transcription and by mRNA stabilisation.

Axin2 is a negative regulator of Wnt/beta-catenin signalling with roles in early development and tumour suppression. Axin2 is induced by E2F1 and therefore acts as a point of cross-talk between the pRb/E2F and Wnt/beta-catenin pathways: two of the most frequently deregulated pathways in human cancers. In this study, we show that E2F1 up-regulates axin2 by two independent mechanisms. The human axin2 gene allows transcription of messages with three different 5' untranslated regions and in the first mechanism E2F1 directly activates the transcription of only one of these species by acting at canonical E2F binding sites. Second, E2F1 induces stabilisation of axin2 mRNAs. We discuss this regulation with respect to other known E2F targets.

5' Untranslated Regions↗

PubMed2006

Identification of novel splice variants of the human CD44 gene.

The CD44 gene contains 10 variable exons (v1-v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms, several of which have been implicated in the metastatic spread of tumour cells. Here, we describe a cryptic splice site, in intron 6 of the human CD44 gene, used during mRNA processing. This cryptic splice site is used in conjunction with variable exon 3, or independently from it in the form of a pseudo-exon of 49 bp, which generates a stop codon by frame shift in the contiguous variable exon downstream. This pseudo-exon has been found inserted immediately 3' to any other variable exon from v4 to v10, in the final CD44 mRNA. The implication of this cryptic splice site in haltering CD44 protein translation is questioned in the context of Nonsense Mediated Decay and the overall regulation of CD44 expression.

Alternative Splicing↗

PubMed2006

The role of endoribonucleases in the regulation of RNase R.

RNase R is an important exoribonuclease involved in the maturation and degradation of RNA. RNase R is co-transcribed with other genes in the same operon. In this report, we show that under physiological conditions maturation of these co-transcripts and the levels of RNase R are mainly dependent on the endoribonuclease RNase E. The presence of the full-length RNase E is necessary for the decay of intermediary products that arise from the maturation of transcripts from the rnr operon. RNase G and RNase III do not seem to have a primary role in the processing of the rnr transcripts. However, the accumulation of intermediary transcripts in an rng mutant suggests that RNase G may act in the degradation of the transcripts already cleaved by RNase E. These results demonstrated that other ribonucleases can act as an additional level of regulation in the control of the expression of RNase R.

Endoribonucleases↗

PubMed2006

Progressive mRNA decay establishes an mkp3 expression gradient in the chick limb bud.

The apical ectodermal ridge (AER) controls limb outgrowth and patterning, such that its removal causes changes in mesodermal gene expression, cell death and limb truncation. Fibroblast growth factor (FGF) family members are expressed in the AER and can rescue limb bud outgrowth after AER removal. Cells localized underneath the AER are maintained in an undifferentiated state by the FGFs produced by the AER. MAPK phosphatase 3 (mkp3) is a downstream effector of FGF8 signalling during limb bud development and is expressed in the distal limb mesenchyme. The present work evidences a gradient of mkp3 transcripts along the chick limb bud, in a distal to proximal direction. mkp3 transcription occurs only in the most distal limb bud cells and its mRNA gradient throughout the limb results from progressive mRNA decay. We show that FGF8-soaked beads induce ectopic mkp3 expression, indicating that AER-derived FGF8 protein may activate mkp3 in the distal mesenchyme.

Animals↗

PubMed2005

Regulation of histone synthesis and nucleosome assembly.

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.

Cell Cycle↗

PubMed2005

Expression of metazoan replication-dependent histone genes.

Histone proteins are essential components of eukaryotic chromosomes. In metazoans, they are produced from the so-called replication-dependent histone genes. The biogenesis of histones is tightly coupled to DNA replication in a stoichiometric manner because an excess of histones is highly toxic for the cell. Therefore, a strict cell cycle-regulation of critical factors required for histone expression ensures exclusive S-phase expression. This review focuses on the molecular mechanisms responsible for such a fine expression regulation. Among these, a large part will be dedicated to post-transcriptional events occurring on histone mRNA, like histone mRNA 3' end processing, nucleo-cytoplasmic mRNA export, translation and mRNA degradation. Many factors are involved, including an RNA-binding protein called HBP, also called SLBP (for hairpin- or stem-loop-binding protein) that binds to a conserved hairpin located in the 3' UTR part of histone mRNA. HBP plays a pivotal role in the expression of histone genes since it is necessary for most of the steps of histone mRNA metabolism in the cell. Moreover, the strict S-phase expression pattern of histones is achieved through a fine cell cycle-regulation of HBP. A large part of the discussion will be centered on the critical role of HBP in histone biogenesis.

Animals↗

PubMed2006

The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements, contains mRNA-instability elements and is involved in glutamine sensing.

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine.

3' Untranslated Regions↗

PubMed2005

SOST is a target gene for PTH in bone.

Intermittent parathyroid hormone (PTH) application is an established pharmacological principle to stimulate bone formation. Yet, the molecular mechanisms underlying this bone anabolic action are not fully understood. Recently, SOST (sclerostin) was identified as a potent osteocyte expressed negative regulator of bone formation in vitro, in murine models and in patients with the bone overgrowth disorders Sclerosteosis and Van Buchem disease. Therefore, we have studied the impact of PTH on SOST regulation. First, we analyzed SOST expression during PTH-induced bone formation in a classical model of local bone formation. 8-month-old mice received intermittently 100 nM hPTH(1-34) or vehicle onto the calvaria for 5 days. PTH stimulated bone formation as assessed by fluorochrome-marker-based histomorphometry. SOST expression was reduced in PTH-treated calvariae 4 h after the last administration as evaluated by real-time quantitative PCR. Next, we observed a decrease of SOST expression in femoral cortical bone of 6-month-old rats following single subcutaneous systemic administration of 80 microg/kg PTH(1-34). Finally, we studied SOST mRNA expression in bone of 11-month-old osteopenic estrogen-deprived (OVX) rats following 8-week systemic intermittent administration of 5 microg/kg PTH(1-34). PTH-treated animals displayed increases in bone mineral density as detected by pQCT, while SOST mRNA levels were decreased compared to vehicle-treated OVX and SHAM controls. PTH decreased SOST expression also in vitro. 100 nM PTH(1-34) inhibited expression in mouse calvaria organ cultures and in osteoblastic UMR-106 cells within 6 h by 95%. An IC50 of 1 nM was determined for PTH(1-34) in UMR-106 cells, whereas the PTH antagonist (d-Trp12,Tyr34)-bPTH(7-34) did not efficiently reduce SOST expression. Furthermore, SOST inhibition by PTH was not blocked by the protein synthesis inhibitor cycloheximide, indicating direct regulation, and PTH did not influence SOST mRNA degradation, implying transcriptional regulation. Finally, we observed full suppression of SOST by the cAMP inducer forskolin, partial inhibition by ionomycin, and no effect with PMA, indicating that PTH action is mainly mediated via the cAMP/PKA pathway. In summary, we have shown that PTH directly inhibits SOST transcription in vivo and in vitro, suggesting that SOST regulation may play a role in mediating PTH action in bone.

Adaptor Proteins, Signal Transducing↗

PubMed2006

RNA regulation and cancer development.

Cancer is viewed as a genetic disease. According to the currently accepted model of carcinogenesis, several consequential mutations in oncogenes or tumor suppressor genes are necessary for cancer development. In this model, mutated DNA sequence is transcribed to mRNA that is finally translated into functionally aberrant protein. mRNA is viewed solely as an intermediate between DNA (with 'coding' potential) and protein (with 'executive' function). However, recent findings suggest that (m)RNA is actively regulated by a variety of processes including nonsense-mediated decay, alternative splicing, RNA editing or RNA interference. Moreover, RNA molecules can regulate a variety of cellular functions through interactions with RNA, DNA as well as protein molecules. Although, the precise contribution of RNA molecules by themselves and RNA-regulated processes on cancer development is currently unknown, recent data suggest their important role in carcinogenesis. Here, we summarize recent knowledge on RNA-related processes and discuss their potential role in cancer development.

Alternative Splicing↗

PubMed2004

Nonsense-mediated mRNA decay: terminating erroneous gene expression.

Nonsense-mediated mRNA decay is a surveillance pathway that reduces errors in gene expression by eliminating aberrant mRNAs that encode incomplete polypeptides. Recent experiments suggest a working model whereby premature and normal translation termination events are distinct as a consequence of the spatial relationship between the termination codon and mRNA binding proteins, a relationship partially established by nuclear pre-mRNA processing. Aberrant termination then leads to both translational repression and an increased susceptibility of the mRNA to multiple ribonucleases.

3' Untranslated Regions↗