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PubMed1992

Viral small nuclear ribonucleoproteins bind a protein implicated in messenger RNA destabilization.

Herpesvirus saimiri (HVS) is one of several primate viruses that carry genes for small RNAs. The five H. saimiri-encoded U RNAs (HSURs) are the most abundant viral transcripts expressed in transformed marmoset T lymphocytes. They assemble with host proteins common to spliceosomal small nuclear ribonucleoproteins (snRNPs). HSURs 1, 2, and 5 exhibit sequences at their 5' ends identical to the AUUUA motif, which targets a number of protooncogene, cytokine, and lymphokine mRNAs for rapid degradation. We show that a 32-kDa protein previously demonstrated to bind to the 3' untranslated region of several unstable messages can be UV crosslinked specifically to HSUR 1, 2, and 5 transcripts in vitro, as well as to endogenous HSUR snRNPs. Our results suggest an unusual role for these viral snRNPs: HSURs may function to attenuate the rapid degradation of certain cellular mRNAs, thereby facilitating viral transformation of host T lymphocytes.

Base Sequence↗

PubMed1993

Synergism in replication and translation of messenger RNA in a cell-free system.

Combination of the Q beta replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recombinant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRNA sequence inserted into RQ135(-1) RNA, an efficient naturally occurring Q beta replicase template. The enhancement is associated with a replication asymmetry previously described for the replication of Q beta phage RNA in vivo; the sense (+)-strands are produced in large excess over the antisense (-)-strands. This, in turn, results in increased synthesis of the functionally active DHFR. These effects are not observed when DHFR mRNAs or RQ135(-1) RNAs are used as templates, if the translation system is not complete, or if it is inhibited by puromycin. The coupled replication-translation of nonviral mRNA recombinants can serve as a useful model for studying the fundamental aspects of virus amplification and can be implemented for large-scale protein synthesis in vitro.

Base Sequence↗

PubMed1996

Length suppression in histone messenger RNA 3'-end maturation: processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA.

Efficient 3'-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem-loop and a histone downstream element (HDE) that base pairs with the U7 small ribonucleoprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Animals↗

PubMed1985

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA.

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.

Anticodon↗

PubMed1984

An improvement on labelled messenger RNA preparation for DNA-RNA hybridisation.

An improved method for the removal of DNA and phenol residues after phenol extraction in m-RNA preparation has been developed. This involves repeated freeze-thaws of the aqueous layer under centrifugal force. As a model assay, five minutes pulse-labelled lambda cI47 late m-RNA is prepared and hybridised to the r-strain lambda DNA. After the centrifugation treatment, non-specific hybridisation is reduced to one-tenth of the untreated sample and the background with respect to input radioactivity for hybridisation is reduced to 0.3%. This low background m-RNA preparation is important especially in dealing with minor class m-RNA.

Bacteriophage lambda↗

PubMed2005

Co-transfection of messenger RNA and siRNA as a method to study the efficiency of siRNA.

The definition of an optimal siRNA results from the in vitro testing of several siRNA designed to specifically target a gene. Usually, such in vitro tests consist in the transfection of the several siRNA duplexes in a cell expressing stably the gene of interest. When a siRNA specific for a mRNA coding toxic proteins (certain transcription factors, transporters, toxins, cell cycle controlling proteins, etc.) must be tested, the generation of a target cell is difficult. Here we report a quick method to test the efficiency of a siRNA through its co-transfection with the targeted mRNA. This technique can be used as a fast method to test siRNA even when they target genes that cannot be stably expressed in the cells of interest.

Animals↗

PubMed1975

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.

Anisomycin↗

PubMed1994

Interactions of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with vesicular stomatitis virus messenger RNA.

The ability of oligonucleotides to interact selectively with their targets is an important consideration in the design of antisense oligonucleotides. This is especially important in the case of antisense oligomers, such as psoralen-derivatized oligomers, which can irreversibly bind to their targets. We have studied the interactions of a series of psoralen-derivatized antisense oligonucleoside methylphosphonates with the mRNAs of vesicular stomatitis virus (VSV), mRNAs that have a high degree of sequence homology. Cross-linking reactions were carried out under conditions of low ionic strength in order to reduce mRNA secondary structure. A 12-mer, whose sequence was complementary to VSV M-mRNA and partially complementary to sequences found in N, NS, and G mRNA cross-linked extensively to N-message. On the other hand, 16-mers whose sequences were uniquely complementary to binding sites on N- or M-mRNA specifically and efficiently cross-linked to their targeted mRNAs over the temperature range 0 degree to 37 degrees C. A reverse transcriptase-catalyzed primer extension assay was used to show that one of the N-specific oligomers cross-linked at the expected site on N-mRNA and to estimate the extent of cross-linking. The results demonstrate that psoralen-derivatized oligonucleoside methylphosphonates can cross-link in a sequence-specific manner if the sequences of these oligomers are chosen carefully so as to avoid extensive partial complementarity with other mRNA sequences.

Animals↗

PubMed1997

Two promoters in expression of estrogen receptor messenger RNA in human breast cancer.

The human estrogen receptor (ER) gene has recently been shown to transcribe two types of mRNA originating from two distinct promoters in mammary tumor cell lines, which encode the same protein. However, use of the two promoters has not been addressed in human breast cancer, which reveals a heterogeneity in terms of ER expression status and clinical characteristics. In this report, we investigated which promoter is responsible for the expression of ER in human mammary tumors by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis for discriminatory detection of the two transcripts in mammary tissues obtained from patients with breast cancer. First, the use of distinct promoters was confirmed in several mammary tumor cell lines by the present RT-PCR method. Secondly, expression levels of total ER mRNA and two types of mRNAs from the different promoters were analysed in tumor, surrounding tissue and normal tissue obtained from 12 patients with breast cancer, which showed various levels of ER protein. In tumors, levels of total ER mRNA and the mRNA transcribed from a distal promoter showed remarkable correlation to the ER protein levels with correlation coefficients 0.946 (P < 0.001) and 0.746 (P < 0.005), respectively. In contrast, mRNA from a proximal promoter showed no correlation to the ER protein levels. Our results indicate that the enhancement of the ER mRNA expression from the distal promoter plays an essential role in the mechanisms of overexpressing ER protein in human mammary tumors, implying that a tumor-specific regulation of ER expression involved use of the distal promoter.

Adult↗

PubMed1998

Non-invasive detection of fecal protein kinase C betaII and zeta messenger RNA: putative biomarkers for colon cancer.

We have developed a non-invasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers. In this study, we used the rat colon carcinogenesis model to determine if changes in fecal protein kinase C (PKC) expression have predictive value in monitoring the neoplastic process. Weanling rats were injected with saline or azoxymethane (AOM) and 36 weeks later fecal samples and mucosa were collected, poly A+ RNA isolated, and quantitative RT-PCR performed using primers to PKC betaII and zeta. Fecal PKC betaII and zeta mRNA levels were altered by the presence of a tumor, with tumor-bearing animals having a 3-fold higher (P < 0.05) PKC betaII expression as compared with animals without tumors. In addition, AOM-injection increased mucosal PKC betaII mRNA expression compared with saline controls. No effect of tumor incidence on mucosal PKC betaII expression was observed. In contrast, fecal PKC zeta expression was 2.5-fold lower (P < 0.05) in animals injected with azoxymethane versus saline. Since tumor incidence exerts a reciprocal effect on fecal PKC betaII and zeta mRNA expression, data were also expressed as the ratio between PKC betaII and zeta. The isozyme ratio was strongly related to tumor incidence, i.e. ratio for animals with tumors was 2.18 +/- 1.25, animals without tumors was 0.50 +/- 0.16, P = 0.025. We demonstrate that the expression of fecal PKC betaII and zeta may serve as a noninvasive marker for development of colon tumors. A sensitive technique for the detection of colon cancer is of importance since early diagnosis can substantially reduce mortality.

Animals↗

PubMed1994

Quantitative immunocytochemical assays of P-glycoprotein in breast carcinomas: correlation to messenger RNA expression and to immunohistochemical prognostic indicators.

BACKGROUND: Chemotherapy failure that is due to cellular drug resistance remains a major problem in most cancer patients. One type of drug resistance that has been characterized is the multidrug resistance phenomenon, which demonstrates a reduced ability of cancer cells to accumulate drugs as a result of the effects of an energy-dependent unidirectional drug efflux pump with a broad substrate specificity. This drug pump is composed of a 170-kd transmembrane glycoprotein referred to as the P-glycoprotein (P-gp) that uses energy in the form of adenosine triphosphate to transport drugs through a channel formed by transmembrane segments. PURPOSE: Our purpose was to detect the levels of P-gp expression in frozen untreated breast carcinomas by immunocytochemical assays and to correlate these levels to current prognostic indicators and, in a few cases, to MDR1 (also known as PGY1) mRNA expression by polymerase chain reaction (PCR). METHODS: The immunocytochemical expression of the multidrug resistance gene, P-gp, was investigated using a specific monoclonal antibody (JSB1) against P-gp in 5-microns frozen sequential sections of breast carcinomas obtained from 213 patients. Microscopic images of immunostained preparations were evaluated by image analysis and were compared with MDR1 transcription (mRNA) assessed by PCR in 16 patients. Quantitative P-gp immunocytochemical assays were correlated to histoprognostic factors and immunocytochemical indicators. RESULTS: Among the 213 breast carcinomas tested, 113 (53%) were P-gp positive, but in 28% of the tumors, the immunostained surface accounted for less than 5% of the total area stained. Quantitative immunocytochemistry reflecting the amount of intracellular P-gp antigen strongly correlated (r = 0.865; two-sided, P < .0001; Pearson's test) with the quantitative evaluation of the scanner analysis of mRNA transcripts. The P-gp expression was significantly (two-sided, P < .001) correlated with p53 expression in tumors, to cathepsin D and Ki67 (two-sided, P < .01) immunoreactivity, and to a lesser extent, the detection of estrogen receptor antigenic sites (two-sided, P = .019). P-gp expression was found to be independent of expression of progesterone receptor and pS2, pHER-2/neu, and CD31 in tumors and from patient age, tumor size, histologic types, grades and Nottingham prognostic index, and nodal status. CONCLUSIONS: The quantitative immunocytochemical assays of P-gp are correlated to PCR analysis of MDR1 expression, and such correlations can be useful in evaluating potential multidrug resistance in breast cancer. However, the clinical significance of P-gp immunodetections remains to be further determined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Differential expression of type II, IV and cytosolic PLA2 messenger RNA in human intrauterine tissues at term.

The involvement of phospholipase A2 (PLA2) enzymes in the formation of biologically-active phospholipid metabolites by human gestational tissues has principally been characterized by the use of enzyme activity assays. While such assays have established the presence of functional PLA2 activity, there is a paucity of information concerning the tissue distribution and relative contribution to net activity made by specific PLA2 isozymes. In particular, both secretory and cytosolic isozymes may be involved in gestational tissue phospholipid metabolism. Thus, the aim of this study was to test the hypothesis that phospholipase A2 mRNA transcripts encoding Type II, Type IV and cytosolic PLA2 are tissue-specifically expressed in human amnion, choriodecidua and placenta obtained at term. The relative expression of polyA+ mRNA encoding these PLA2 isozymes was determined by Northern blot analysis and laser densitometry. The data obtained confirm the tissue-specific expression of PLA2 mRNA in human intrauterine tissues. Cytosolic PLA2 mRNA was most abundantly expressed in amnion when compared to either choriodecidua (which was 5-fold less than amnion; P < 0.001) or placenta (72-fold less than amnion; P < 0.001). In contrast, the secretory PLA2 mRNA transcripts (i.e. Type II and Type IV) were most abundantly expressed in placenta. Type II PLA2 mRNA expression in choriodecidua and amnion was 30-fold less than that observed in placenta (both P < 0.001). Type IV PLA2 mRNA expression was 37-fold (P < 0.001) and 73-fold (P < 0.001) less in choriodecidua and amnion respectively. These data support the conclusion that cytosolic PLA2 is the principal PLA2 isozyme mediating phospholipid metabolism and the liberation of fatty acid substrate (i.e. arachidonic acid) in term amnion, while secretory PLA2 isozymes, and in particular, Type II PLA2 play a major role in phospholipid metabolism in term placenta.

Amnion↗

PubMed1988

The human U1 snRNA promoter and enhancer do not direct synthesis of messenger RNA.

We examined the ability of the 5' flanking region sequences of a human U1 RNA gene to direct synthesis of functional mRNA. When fused to chloramphenicol acetyltransferase (CAT) coding region sequences, the upstream sequences of the U1 gene were able to stimulate the synthesis of functional CAT mRNA in 293 cells but not in HeLa cells. Most of the polyadenylated CAT mRNA in 293 cells originated from cryptic promoters in the upstream U1 sequences, but nearly all of the CAT-specific RNA originating at position +1 (relative to the U1 gene promoter) was non-polyadenylated; this confirmed that the bona-fide U1 gene promoter was unable to direct efficient synthesis of poly-A+ mRNA. Our results demonstrate that the snRNA gene promoter and enhancer elements, although very efficient in transcription of snRNAs, are unable to direct transcription of polyadenylated mRNAs. However, other sequences in the 5' flanking region of the human U1 gene can activate transcription of functional mRNA, with 5' ends upstream of the normal transcription start site.

Acetyltransferases↗

PubMed1975

m7G5'ppp5'GmptcpUp at the 5' terminus of reovirus messenger RNA.

In the presence of S-adenosyl methionine the 5' terminal guanosine residue of in vitro synthesized reovirus mRNA becomes methylated at the 2'-OH position. In addition, 7-methyl guanylic acid is condensed covalently at the 5' terminus resulting in the formation of a 5' to 5' triphosphate bridge. Analysis of the 5' terminal sequence of methylated reovirus mRNA revealed that it has the structure m7G5'ppp5'GmpCpUp.

Base Sequence↗

PubMed1992

Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.

Amino Acid Sequence↗

PubMed1997

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

Animals↗

PubMed1997

Specific polyadenylation and purification of total messenger RNA from Escherichia coli.

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Blotting, Northern↗

PubMed1976

The isolation and characterization of bacteriophage T7 messenger RNA fragments containing an RNase III cleavage site.

We have isolated overlapping RNA fragments which contain the region surrounding the ribonuclease III cleavage site between bacteriophage T7 genes 0.3 and 0.7. Although all of these fragments contain the site of cleavage, only certain fragments are correctly recognized and cleaved by RNase III. Analysis of the cleavage products of the fragments indicates that the enzyme produces a single endonucleolytic break at this site in the T7 early RNA precursor molecule. In addition, the 3'-terminal adenylic acid residues observed previously on the in vivo T7 early RNA species were not found in these fragments and, therefore, must represent a post-transcriptional, post-processing modification of the RNA.

Base Sequence↗