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PubMed1980

Suppression of murine retrovirus polypeptide termination: effect of amber suppressor tRNA on the cell-free translation of Rauscher murine leukemia virus, Moloney murine leukemia virus, and Moloney murine sarcoma virus 124 RNA.

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200(gag-pol) polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65(gag) and Moloney murine leukemia virus Pr63(gag). Under suppressor-minus conditions, Moloney murine leukemia virus Pr70(gag) was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the "upper" Moloney murine leukemia virus Pr70(gag) polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200(gag-pol) coincided closely with the molar loss of Pr63(gag). Enhancement of Pr200(gag-pol) and Pr70(gag) by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63(gag) as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63(gag), P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67(gag), appeared, whereas Pr63(gag) synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67(gag) which appeared, 1 mol of Pr63(gag) was lost.

Kinetics↗
PubMed2006

An enhanced RNA alignment benchmark for sequence alignment programs.

BACKGROUND: The performance of alignment programs is traditionally tested on sets of protein sequences, of which a reference alignment is known. Conclusions drawn from such protein benchmarks do not necessarily hold for the RNA alignment problem, as was demonstrated in the first RNA alignment benchmark published so far. For example, the twilight zone - the similarity range where alignment quality drops drastically - starts at 60 % for RNAs in comparison to 20 % for proteins. In this study we enhance the previous benchmark. RESULTS: The RNA sequence sets in the benchmark database are taken from an increased number of RNA families to avoid unintended impact by using only a few families. The size of sets varies from 2 to 15 sequences to assess the influence of the number of sequences on program performance. Alignment quality is scored by two measures: one takes into account only nucleotide matches, the other measures structural conservation. The performance order of parameters--like nucleotide substitution matrices and gap-costs--as well as of programs is rated by rank tests. CONCLUSION: Most sequence alignment programs perform equally well on RNA sequence sets with high sequence identity, that is with an average pairwise sequence identity (APSI) above 75 %. Parameters for gap-open and gap-extension have a large influence on alignment quality lower than APSI < or = 75 %; optimal parameter combinations are shown for several programs. The use of different 4 x 4 substitution matrices improved program performance only in some cases. The performance of iterative programs drastically increases with increasing sequence numbers and/or decreasing sequence identity, which makes them clearly superior to programs using a purely non-iterative, progressive approach. The best sequence alignment programs produce alignments of high quality down to APSI > 55 %; at lower APSI the use of sequence+structure alignment programs is recommended.

Journal Article↗
PubMed2005

Resolution enhanced homonuclear carbon decoupled triple resonance experiments for unambiguous RNA structural characterization.

Large RNAs (>30 nucleotides) suffer from extensive resonance overlap that can seriously hamper unambiguous structural characterization. Here we present a set of 3D multinuclear NMR experiments with improved and optimized resolution and sensitivity for aiding with the assignment of RNA molecules. In all these experiments strong base and ribose carbon-carbon couplings are eliminated by homonuclear band-selective decoupling, leading to improved signal to noise and resolution of the C5, C6, and C1' carbon resonances. This decoupling scheme is applied to base-type selective 13C-edited NOESY, 13C-edited TOCSY (HCCH, CCH), HCCNH, and ribose H1C1C2 experiments. The 3D implementation of the HCCNH experiment with both carbon and nitrogen evolution enables direct correlation of 13C and 15N resonances at different proton resonant frequencies. The advantages of the new experiments are demonstrated on a 36 nucleotides hairpin RNA from domain 5 (D5) of the group II intron Pylaiella littoralis using an abbreviated assignment strategy. These four experiments provided additional separation for regions of the RNA that have overlapped chemical shift resonances, and enabled the assignment of critical D5 bulge nucleotides that could not be assigned using current experimental schemes.

Carbon Isotopes↗
PubMed2003

Immune-enhancing diets for stressed patients with a special emphasis on arginine content: analysis of the analysis.

PURPOSE OF REVIEW: After two decades of intensive research, whether arginine-enriched diets for oral/enteral administration are beneficial or harmful for stressed patients remains uncertain. An American consensus and a meta-analysis provide divergent conclusions. The main goal of the present review is to analyze these documents. RECENT FINDINGS: The so-called immune-enhancing diets have been found to be beneficial to postoperative patients. Nothing proves, however, that arginine is responsible for these beneficial effects since immune-enhancing diets contain other pharmacologically active components (e.g. omega3 free fatty acids, RNAs, antioxidant vitamins). In fact, arginine-enriched diets may be harmful in hemodynamically unstable patients and those presenting with multiple organ failure. SUMMARY: In light of the current doubts and until convincing data are produced, immune-enhancing diets should not be used in unstable critically ill patients.

Adjuvants, Immunologic↗
PubMed1994

Nuclear export of different classes of RNA is mediated by specific factors.

Various classes of RNA are exported from the nucleus to the cytoplasm, including transcripts of RNA polymerase I (large ribosomal RNAs), II (U-rich small nuclear RNAs [U snRNAs], mRNAs), and III (tRNAs, 5S RNA). Here, evidence is presented that some steps in the export of various classes of nuclear RNA are mediated by specific rather than common factors. Using microinjection into Xenopus oocytes, it is shown that a tRNA, a U snRNA, and an mRNA competitively inhibit their own export at concentrations at which they have no effect on the export of heterologous RNAs. While the export of both U snRNAs and mRNAs is enhanced by their 7-methyl guanosine cap structures, factors recognizing this structure are found to be limiting in concentration only in the case of U snRNAs. In addition to the specific factors, evidence for steps in the export process that may be common to at least some classes of RNA are provided by experiments in which synthetic homopolymeric RNAs are used as inhibitors.

Animals↗
PubMed1980

Initiation of reovirus transcription by inosine 5'-triphosphate and properties of 7-methylinosine-capped, inosine-substituted messenger ribonucleic acids.

Inosine 5'-triphosphate (ITP) can be utilized in place of guanosine 5'-triphosphate (GTP) for both the initiation and the elongation steps of reovirus transcription, resulting in the synthesis of mRNAs containing 5'-terminal m7IpppIm and internal pI. The apparent molecular weights of the I-substituted products were altered as a consequence of the absence of G-C base pairs and accompanying loss of ordered structure. The migration of I-substituted RNAs in agarose gels and glycerol gradients was similar to glyoxal-treated transcripts; i.e., it decreased 2-fold as compared to the corresponding untreated G-containing mRNAs. 7-Methylinosine-capped (m7I-capped), I-substituted transcripts readily attached to wheat germ 80S ribosomes. Unlike native G-containing mRNAs, they also formed heavier complexes that sedimented faster than 80S complexes even in the presence of the nonhydrolyzable ATP analogue AMPP(NH)P and elongation inhibitor sparsomycin. I-substituted molecules that were capped posttranscriptionally to form m7G-capped 5' ends yielded mostly 80S monosomes, consistent with a strong influence of 5'-terminal structure on initiation of translation. Under limited conditions of initiation, I-substituted RNAs outcompeted G-containing transcripts for ribosome attachment. Although the results are consistent with enhanced binding and freer movement of ribosomes on unstructured templates, synthesis of acid-precipitable polypeptides in wheat germ extract directed by I-substituted RNAs was 15-fold less than with G-containing mRNAs.

Binding Sites↗
PubMed2003

Identification of a gadd45beta 3' enhancer that mediates SMAD3- and SMAD4-dependent transcriptional induction by transforming growth factor beta.

GADD45beta regulates cell growth, differentiation, and cell death following cellular exposure to diverse stimuli, including DNA damage and transforming growth factor-beta (TGFbeta). We examined how cells transduce the TGFbeta signal from the cell surface to the gadd45beta genomic locus and describe how GADD45beta contributes to TGFbeta biology. Following an alignment of gadd45beta genomic sequences from multiple organisms, we discovered a novel TGFbeta-responsive enhancer encompassing the third intron of the gadd45beta gene. Using three different experimental approaches, we found that SMAD3 and SMAD4, but not SMAD2, mediate transcription from this enhancer. Three lines of evidence support our conclusions. First, overexpression of SMAD3 and SMAD4 activated the transcriptional activity from this enhancer. Second, silencing of SMAD protein levels using short interfering RNAs revealed that TGFbeta-induced activation of the endogenous gadd45beta gene required SMAD3 and SMAD4 but not SMAD2. In contrast, we found that the regulation of plasminogen activator inhibitor type I depended upon all three SMAD proteins. Last, SMAD3 and SMAD4 reconstitution in SMAD-deficient cancer cells restored TGFbeta induction of gadd45beta. Finally, we assessed the function of GADD45beta within the TGFbeta response and found that GADD45beta-deficient cells arrested in G2 following TGFbeta treatment. These data support a role for SMAD3 and SMAD4 in activating gadd45beta through its third intron to facilitate G2 progression following TGFbeta treatment.

Animals↗
PubMed1997

Evaluating and enhancing ribozyme reaction efficiency in mammalian cells.

The ability of ribozymes to cleave specific transcripts and repair defective RNAs in the test tube has engendered speculation about their potential clinical utility. Therapeutic development has been hindered by an inability to evaluate and optimize the efficiency of RNA catalysis in vivo. We describe an experimental system that has allowed us to assess and enhance the efficiency with which a trans-splicing group I ribozyme reacts with a targeted RNA in mammalian cells. These results demonstrate that the ribozyme can convert up to 49% of a specific substrate RNA to product in the cellular environment and that the efficiency of this reaction is apparently a function of the ribozyme's ability to find and bind to the substrate RNA in the cell. These observations suggest that trans-splicing ribozymes may become useful reagents to repair a therapeutically significant fraction of mutant RNAs associated with a variety of genetic diseases.

Animals↗
PubMed2005

Cationic lipids enhance siRNA-mediated interferon response in mice.

RNA interference mediated by small interfering RNAs (siRNAs) shows promise as a powerful research tool for gene function studies. However, controversy exists over the potential of siRNA-induced interferon response in vitro and in vivo. In this study, we showed that although intravenous administration of siRNA alone is essentially inert, injection of siRNA complexed with cationic liposomes resulted in a potent induction of both type I and type II interferon responses. Furthermore, i.v. administration of cationic lipid/siRNA complexes led to activation of STAT1. This study suggests caution in data interpretation and the potential toxicity with in vivo use of siRNA, particularly when delivered via a cationic lipid vector. This study also suggests the potential of siRNA as an immunostimulatory agent for immunotherapy.

Animals↗
PubMed2004

Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells.

Double-stranded short interfering RNAs (siRNAs) mediate post-transcriptional inhibition of gene expression in a variety of biological systems. However, human liver cells show poor uptake of these nucleic acids. In order to improve the delivery of siRNA into these cells without transfection agents, we have synthesized two series of lipophilic siRNAs conjugated with derivatives of cholesterol, lithocholic acid or lauric acid. The lipid moieties were covalently linked to the 5'-ends of the RNAs using phosphoramidite chemistry. The potency of these chemically modified siRNAs to inhibit reporter gene expression was further investigated in vitro with beta-galactosidase expressing liver cells.

Cholesterol↗
PubMed2003

Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependant manner.

The RegB protein, encoded by the T4 bacteriophage genome, is a ribonuclease involved in the inactivation of the phage early messenger RNAs. Its in vitro activity is very low but can be enhanced up to 100-fold in the presence of the ribosomal protein S1. The latter is made of six repeats of a conserved module found in many other proteins of RNA metabolism. Considering the difference between its size (556 amino acids) and that of several RegB substrates (10 nucleotides), we wondered whether all six modules are necessary for RegB activation. We studied the influence of twelve S1 fragments on the cleavage efficiency of three short substrates. RegB activation requires the cooperation of different sets of modules depending on the substrates. Two RNAs are quite well cleaved in the presence of the fragment formed by the fourth and fifth modules, whereas the third requires the presence of the four C-terminal domains. However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or tetra-modules are similar, suggesting a common interaction surface. In the case of the tetra-module the interactions involve all four domains, raising the question of the spatial organization of this region.

Amino Acid Sequence↗
PubMed1984

Differences in physical and biological properties of 50S ribosomes and 23S RNAs derived from tight and loose couple 70S ribosomes.

Tight couple (TC) 50S ribosomes on treatment with kethoxal lose their capacity to associate with 30S ribosomes whereas loose couple (LC) 50S ribosomes on such treatment fully retain their association capacity. The same is true for 23S RNAs isolated from treated 50S ribosomes or isolated 23S RNAs directly treated with kethoxal, so far as their capacity to associate with 16S RNA is concerned. At certain Mg++ concentrations TC 23S RNA is highly susceptible to the nucleolytic action of single-strand specific enzyme RNase I; LC 23S RNA is quite resistant. The Mg++-dependencies of the two species of 23S RNAs for association with 16S RNA are also quite different. The fluorescence enhancement of ethidium bromide due to binding to TC 23S RNA is slightly less than LC 23S RNA. The hyperchromicity of LC 23S RNA due to thermal denaturation is somewhat more than TC 23S RNA. LC 23S RNA has slightly more elliptic CD spectrum than TC 23S RNA. These results clearly show that 23S RNAs present in TC and LC 50S ribosomes are distinct from each other. It has been recently demonstrated in this laboratory that they can be interconverted by the agents involved in translocation and thus appear to be conformomers.

Aldehydes↗
PubMed1996

Role of polyadenylation in nucleocytoplasmic transport of mRNA.

To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3' terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozyme cleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to serve export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a long stretch of poly(A) near the 3' end of a ribozyme-cleaved transcript is not sufficient for directing mRNA export. Taken together, these results suggest that polyadenylation is required for the nucleocytoplasmic transport of mRNA and that Rev interaction with the Rev-responsive element cannot bypass this requirement.

Base Sequence↗
PubMed1996

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.

Adenoviridae↗
PubMed1983

Regulation of gene expression in corn (Zea mays L.) by heat shock. II. In vitro analysis of RNAs from heat-shocked seedlings.

Five-day-old maize seedlings subjected to heat shock exhibit a dramatic enhancement in the synthesis of a small group of polypeptides. Isolation of total RNA from control and heat-shocked maize plumules, fractionation of poly(A)+ mRNA by oligo(dT)-cellulose chromatography, and in vitro translations of the RNAs in both the rabbit reticulocyte and the wheat germ systems indicates that there is remarkable fidelity of the mRNA pool obtained from heat-shocked plumules to reproduce in vitro those same polypeptides whose synthesis is greatly elevated in the intact, heat-shocked plumule. Moreover, these heat-shock polypeptides with molecular masses of 108 000, 89 000, 84 000, 73 000, and 18 000 are translated from polyadenylated mRNAs. The absence of a 76 000 dalton heat-shock polypeptide (HSP) and the presence of fewer isoelectric point variants of the 89 000 and 84 000 dalton HSPs among the in vitro translation products suggests that translational and (or) posttranslational regulatory mechanisms might be operative in determining the final spectrum of the maize heat-shock proteins.

Animals↗
PubMed1988

Alternative use of a polyadenylation signal and of a downstream 3' splice site. Effect of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site P1 instead of the major promotor-distal sites P2 and P4. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of P1. The nascent transcript is first cleaved in the branch point-3' splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3' end (F-RNA) and a promoter-distal RNA with a heterogeneous 5' end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3' end of F-RNA is about 130 nucleotides downstream from P1 and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CP1, which is the transcript ending at P1. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3' end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.

Adenoviridae↗
PubMed2025

Role of lncRNA PVT1 in the progression of urological cancers: Novel insights into signaling pathways and clinical opportunities.

Urologic malignancies, encompassing cancers of the kidney, bladder, and prostate, represent approximately 25&#xa0;% of all cancer cases. Recent advances have enhanced our understanding of PVT1's crucial functions. Long noncoding RNAs influence both the onset and development of cancer, as well as epigenetic alterations. Recent findings have focused on PVT1's mechanism of action across several malignancies, particularly urologic cancers. Understanding the various functions of PVT1 linked to cancer is necessary for the development of cancer detection and treatment when PVT1 is dysregulated. Furthermore, recent advancements in genomic and epigenetic research have elucidated the complex regulatory networks that control PVT1 expression. Comprehending the intricate role of PVT1 Understanding the complex function of PVT1 in urologic cancers has substantial clinical implications. Here, we summarize some of the most recent findings about the carcinogenic effects of PVT1 signaling pathways and the possible treatment strategies for urological malignancies that target these pathways.

Humans↗
PubMed2004

Epigenetic regulation of mammalian genomic imprinting.

Imprinted genes play important roles in development, and most are clustered in large domains. Their allelic repression is regulated by 'imprinting control regions' (ICRs), which are methylated on one of the two parental alleles. Non-histone proteins and nearby sequence elements influence the establishment of this differential methylation during gametogenesis. DNA methylation, histone modifications, and also polycomb group proteins are important for the somatic maintenance of imprinting. The way ICRs regulate imprinting differs between domains. At some, the ICR constitutes an insulator that prevents promoter-enhancer interactions, when unmethylated. At other domains, non-coding RNAs could be involved, possibly by attracting chromatin-modifying complexes. The latter silencing mechanism has similarities with X-chromosome inactivation.

Animals↗