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PubMed1983

Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.

Using a whole cell extract of HeLa cells, we synthesized unspliced RNAs containing the first two leaders and the first intervening sequence of the adenovirus 2 major late transcription unit. Upon incubation of these pre-mRNAs in reaction mixtures containing a nuclear extract and a postnuclear fraction (S100), removal of the first intervening sequence and concomitant joining of the first leader to the second leader was observed. This splicing reaction requires proteins, Mg2+ ions, and ATP. The S100 fraction alone has no splicing activity but stimulates splicing when added to the nuclear extract. Upon fractionation of the postnuclear S100 by chromatography on ion exchange and gel filtration columns, the stimulatory activity copurifies with small ribonucleoprotein particles.

Adenosine Triphosphate↗
PubMed1983

Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein.

A mouse monoclonal antibody and human autoimmune sera directed against various classes of small ribonucleoprotein particles have been tested for inhibition of mRNA splicing in a soluble in vitro system. The splicing of the first and second leader exons of adenovirus late RNA was inhibited only by those sera that reacted with U1 RNP. Both U1 RNP-specific human autoimmune serum and sera directed against the Sm class of small nuclear RNPs, including a mouse monoclonal antibody, specifically inhibited splicing. Antisera specific for U2 RNP had no effect on splicing nor did antisera specific for the La or Ro class of small RNPs. These results suggest that U1 RNP is essential for the splicing of mRNA precursors.

Adenoviruses, Human↗
PubMed1985

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors.

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.

Base Sequence↗
PubMed1981

Ribosomal and translatable messenger RNA of Eimeria tenella.

RNA from oocysts of Eimeria tenella was purified into poly(A)--RNA and poly(A)+-RNA fractions with affinity chromatography on oligo(dT)-cellulose. Gel electrophoresis, under denaturing conditions of the poly(A)--RNA fraction revealed two major RNAs with molecular weights of 1.27 and 0.65 X 10(6). The values for these components not only represent the ribosomal RNAs of this species; but they also resemble the molecular sizes of the non-mRNAs of many other lower eukaryotes. In vitro translation assays with a cell-free wheat germ system showed that the level of translatable mRNA in the poly(A)--RNA fraction was negligible. Virtually all the translatable mRNA in E. tenella was polyadenylated, that is, it contained poly(A)-tracts with more than 15 adenylate residues. Consequently some of the key components of the protein translational machinery of this organism are eukaryotic in nature.

Animals↗
PubMed1982

Efficient extraction and translation of Plasmodium falciparum messenger RNA.

A convenient method was employed for the efficient extraction of total RNA from Plasmodium falciparum. By depleting the total RNA of tRNA, it was shown that P. falciparum or P. lophurae tRNAs markedly stimulate the translation of P. falciparum mRNA in the rabbit reticulocyte cell-free system. Analysis of the cell-free products revealed the presence of proteins well over 200 000 molecular weight, with the majority of polypeptides having high molecular weights. Thus, this system can now be used to isolate and characterize specific mRNA molecules from P. falciparum.

Animals↗
PubMed1982

The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae.

Preliminary studies had indicated the inadequacy of the wheat germ and rabbit reticulocyte cell-free translation systems for the in vitro translation of mRNA isolated from Plasmodium lophurae. To identify the factors which are important for the efficient translation of parasite proteins, an homologous system was established using polysomes, the pH 5 fraction, and tRNA prepared from p. lophurae. For comparison, the same components were isolated from the host duck reticulocytes and tested. The effect of each of these factors was evaluated by analysis of the translation products and by comparison with products synthetized in vivo. The results indicated that P. lophurae tRNA had a marked stimulatory effect on the synthesis of parasite proteins while it inhibited the synthesis of host proteins. Duck reticulocyte tRNA could not be used as a substitute for the parasite tRNA. Based on these findings, a commercially available rabbit reticulocyte system was supplemented with P. lophurae tRNA, which markedly increased the efficiency of translation of P. lophurae proteins by this system.

Animals↗
PubMed1992

Binding of ATP and messenger RNA by the beta-subunit of eukaryotic initiation factor 2.

In addition to forming a ternary complex with Met-tRNA(f) and GTP, eukaryotic initiation factor 2 (eIF-2) recognizes a specific site in mRNA molecules. Both binding activities are regulated by ATP, which itself binds tightly and specifically to eIF-2. Denaturation of eIF-2 with urea leads to complete loss of Met-tRNA(f) binding activity, while mRNA binding activity is stable. Hence, distinct conformational features in eIF-2 are required for ternary complex formation and for binding of mRNA. Chromatography of eIF-2 over ATP-agarose, in denaturing conditions that induce polypeptide subunit dissociation, results in selective retention of the beta-subunit of eIF-2. Isolated beta-subunit is capable of binding mRNA as well as ATP. Cibacron blue 3G-A binds tightly to eIF-2 and inhibits the binding of mRNA. This inhibition is relieved upon addition of ATP, showing that Cibacron blue 3G-A competes with ATP for eIF-2. eIF-2 beta subunit, active in binding of mRNA, is recovered upon chromatography of eIF-2 in denaturing conditions over matrix-bound Cibacron blue 3G-A. These results show that the ability of eIF-2 to bind mRNA and its ability to bind ATP are both lodged within remarkably stable domains of its beta-subunit. During initiation of protein synthesis, the eIF-2 beta subunit may thus interact with three ligands important for translational control: Met-tRNA(f), mRNA and ATP.

Adenosine Triphosphate↗
PubMed1975

Possible mechanism of E. coli messenger RNA degration: non-enzymatic degradation.

The present paper points out lack of evidence to support the presently prevailing concept that E. coli mRNA turnover, in the gene expression process, cannot take place without mRNase(s). The present paper draws attention to possible physicochemical factors involved in the degradation, and advances a notion of non-enzymatic spontaneous degradation of E. coli mRNA in its expression process. This suggested hypothesis helps to explain hitherto reported findings on the mode of E. coli mRNA degradation.

Biodegradation, Environmental↗
PubMed1985

Translational interference at overlapping reading frames in prokaryotic messenger RNA.

In overlapping reading frames of prokaryotic mRNA, the ribosome-binding site (RBS) of the downstream cistron is part of the coding sequence of the upstream message. We have examined whether the rate of translation in Escherichia coli can be sufficiently high to preclude the use of an RBS in initiation of protein synthesis when it is part of an actively decoded reading frame. The two sets of gene overlap present in the RNA phage MS2 are used as a model system. We find that translation of an upstream cistron can fully block initiation of protein synthesis at the overlapping RBS of the downstream cistron. Nonsense mutations in the upstream gene restore the translation of the downstream gene.

Binding Sites↗
PubMed1989

Nucleotide sequence of the 24S subgenomic messenger RNA of a vaccine strain (HPV77) of rubella virus: comparison with a wild-type strain (M33).

A full-length cDNA clone for the 24S subgenomic mRNA of the vaccine strain (HPV77) of rubella virus has been isolated from a cDNA library made from the RNAs of infected cells. Starting from the first Met start codon, the 24S mRNA codes for a precursor protein of 1063 amino acids (aa). This precursor encodes a capsid protein of 300 aa, and two envelope proteins, E1 (481 aa) and E2 (282 aa). Both the E1 and E2 proteins are preceded by a stretch of 21 hydrophobic aa, characteristic of a signal peptide, and each has three putative glycosylation sites in the polypeptide chains. Comparison between the structural proteins of the vaccine and the wild-type (wt; M33) strains of rubella virus, revealed that the E2 protein of the vaccine strain differs, in its apparent Mr, by approx. 3 kDa, from the wt strain. The difference could be due to decreased glycosylation of the vaccine strain E2 protein, as revealed by [3H]mannose incorporation studies. Five single-aa changes in the structural proteins occurred during the attenuation process, one each in the capsid and the E1 protein and three in the E2 protein. The change of Thr-412----Ile in the E2 protein results in the loss of a putative glycosylation site at Asn-410, which offers a plausible explanation for decreased glycosylation of the E2 protein from the vaccine strain of rubella virus.

Amino Acid Sequence↗
PubMed1996

Multiple drug resistance-related messenger RNA expression in archival formalin-fixed paraffin-embedded human breast tumour tissue.

A method is described by which RNA, suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, can be extracted from formalin-fixed paraffin-embedded (FFPE) tissues and subsequently used for detecting the expression of several genes. Using this technique, RNA can be extracted from specimens, quantified, reverse transcribed and regions of interest amplified and analysed within 36 h. The tissue specimens included in this study were from human breast carcinoma, investigating a range of genes associated with the development and/or maintenance of multiple drug resistance (MDR). This technique, applied to archival tissues, offers great potential for increasing our understanding of alterations in expression levels of genes associated with MDR. The method developed is also applicable to studies on expression of other genes in paraffin-embedded tissues.

Adult↗
PubMed1993

Wild type and alternatively spliced estrogen receptor messenger RNA in human meningioma tissue and MCF7 breast cancer cells.

Human meningiomas are rich in progesterone receptors (PR), which appear to be expressed autonomously. To investigate whether estrogen receptor (ER) variants which do not bind the ligand, but may constitutively induce PR expression, prevail in meningioma, we amplified cDNA by PCR in order to detect mRNA coding for the ER in meningioma which were ER-negative/PR-positive at the protein level. We screened for a portion of the ER which includes the DNA binding domain, the hinge region and the ligand binding domain. For this part of the ER we found a wild type mRNA in all 8 meningiomas tested. No mutations were detected. Apart from this transcript we found two alternatively spliced products missing exons 4 and 7, respectively in 8/8 meningioma specimens. These two products were not exclusive for meningioma, since they were also detected in the MCF7 breast cancer cell line which was used as control. ER deletion mutants missing exon 7 have already been reported [Ref. 1; Molec. Endocr. 5 (1991) 1571-1577]. These are dominant negative. To our knowledge, this is the first report on ER mutants missing exon 4. The presence of ER variants missing exon 4, which is probably not able to bind heat shock protein 90 and therefore may be constitutively active, might explain the autonomous expression of PR in meningioma.

Alternative Splicing↗
PubMed2005

Splice variant-specific silencing of angiotensin II type 1a receptor messenger RNA by RNA interference in vascular smooth muscle cells.

In the rat, two distinct angiotensin II type 1a (rAT(1a)) receptor mRNAs are synthesized from a single rAT(1a) receptor gene by alternative splicing. These two transcripts are comprised of exons 1, 2, and 3 (E1,2,3) or exons 1 and 3 (E1,3). Since exon 3 contains the entire coding region, both transcripts encode identical rAT(1a) receptors. Real-time PCR revealed that in rat aortic smooth muscle cells (RASMC), E1,2,3 mRNA accounted for 69.5+/-0.9% of total rAT(1a) receptor mRNA. The aim of this study was to use RNA interference (RNAi) to selectively silence the rAT(1a) receptor splice variants. Forty-eight hour treatment of RASMC with E1,3-targeting siRNA (10nM; S1(E1,3)) resulted in a 91.2+/-0.5% (n=3, P<0.001) reduction in E1,3 mRNA and a 19.0+/-3.0% (n=4, P<0.05) reduction in AT(1) receptor specific binding compared with cells treated with a non-silencing control siRNA; under these conditions, no effect was observed on levels of E1,2,3 mRNA. Conversely, treatment with E1,2,3-targeting siRNA (S2(E2)) had no effect on E1,3 mRNA while reducing E1,2,3 mRNA by 73.9+/-4.2% (n=3, P<0.001), and AT(1) receptor binding by 39.4+/-5.4% (n=4, P<0.001) compared with control. These data show that the majority of functional AT(1) receptor expression in RASMC derives from the E1,2,3 splice variant. These data also demonstrate that rAT(1a) receptor mRNA can be silenced in a splice-variant specific manner using siRNA in RASMC, thus providing an excellent model system for investigating the role of alternative splicing in the regulation of rAT(1a) receptor expression.

Alternative Splicing↗
PubMed2005

tmRNA-induced release of messenger RNA from stalled ribosomes.

A ribosome stalled on a truncated mRNA in the eubacterial cell can be rescued by tmRNA via a process called trans-translation. We demonstrate here that release of truncated mRNAs from stalled ribosomes accelerates significantly already after trans-peptidation following tmRNA binding to the ribosome. However, rapid release of truncated mRNA requires EF-G-dependent translocation of peptidyl-tmRNA from the A to the P site of the ribosome. We show also that the rate of mRNA release before and after peptidyl-tmRNA translocation correlates well with the rate of dissociation of deacylated tRNA, indicating that mRNA is retained on the ribosome mainly through codon:anticodon interaction with tRNA. The rate of mRNA release is reduced for mRNAs with strong Shine-Dalgarno (SD)-like sequences in the vicinity of the truncation site as well as for mRNAs with long 3' extensions downstream from the P-site codon. The reduced rate of release in the former case was due to a persisting SD-anti SD interaction between mRNA and the ribosome.

Binding Sites↗
PubMed2005

Messenger RNA processing sites in Trypanosoma brucei.

In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .

5' Untranslated Regions↗
PubMed2004

Messenger RNA stability in mitochondria: different means to an end.

Gene expression is regulated at many stages not merely at the level of transcription. Among the important post-transcriptional processes, RNA turnover has a crucial role. The stability of mRNA in the cytosol of eukarya is increased by the addition of a 3' poly(A) extension. By contrast, this process mediates rapid RNA decay in prokarya. How is mRNA turnover regulated in mitochondria? Their monophyletic, alpha-proteobacterial origin predicts that polyadenylation will induce rapid decay by nucleases and associated factors that are similar to their bacterial ancestors. In this article, however, we report that the regulation of mitochondrial mRNA turnover in diverse species is surprisingly different.

Animals↗
PubMed2003

Full-length telomerase reverse transcriptase messenger RNA is an independent prognostic factor in neuroblastoma.

Telomerase activity (TA) is the most recently recognized prognostic factor in neuroblastoma, and its outstanding predictive power was documented by several studies. However, TA measurements require fresh tumor tissue that is not always available in daily clinical practice. We previously described a reverse transcriptase-polymerase chain reaction assay that we used to investigate the possible prognostic relevance of the telomerase catalytic subunit, hTERT, at the mRNA level. Because hTERT mRNA undergoes alternative splicing as a regulatory mechanism of TA, we discriminated between truncated and full-length hTERT transcripts. In a retrospective study on 124 neuroblastomas, 56 (45.2%) tumors showed spliced hTERT transcripts, whereas 30 (24.2%) contained full-length hTERT transcripts. The presence of both spliced and full-length hTERT transcripts was significantly associated with MYCN amplification. hTERT in general showed no correlation to other prognostic factors, ie, International Neuroblastoma Staging System stage, International Neuroblastoma Pathology classification grade, or age at diagnosis, whereas the presence of full-length transcripts was significantly associated with higher stages. The presence of any hTERT transcripts carried no significant prognostic information, yet full-length hTERT transcripts were highly predictive of poor outcome (P < 0.0001). In a multivariate analysis, full-length hTERT transcripts and International Neuroblastoma Pathology classification grade emerged as the sole independent predictors of event-free survival, with relative risks of 10.0 and 3.9, respectively. The strong statistical correlation of full-length hTERT transcripts with clinical outcome in neuroblastoma suggests that the reverse transcriptase-polymerase chain reaction analysis of hTERT transcripts may be equatable to TA measurements. Because this assay is well suited for archival material, it could become a useful adjunct in evaluating the prognosis of individual neuroblastoma cases.

Brain Neoplasms↗