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At least 775 records · Page 43Linked to original sources

PubMed2001

Probing the folding landscape of the Tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway.

Large, structured RNAs traverse folding landscapes in which intermediates and long-lived misfolded states are common. To obtain a comprehensive description of the folding landscape for a structured RNA, it is necessary to understand the connections between productive folding pathways and pathways to these misfolded states. The Tetrahymena group I ribozyme partitions between folding to the native state and to a long-lived misfolded conformation. Here, we show that the observed rate constant for commitment to fold to the native or misfolded states is 1.9 min(-1) (37 degrees C, 10 mM Mg(2+)), the same within error as the rate constant for overall folding to the native state. Thus, the commitment to alternative folding pathways is made late in the folding process, concomitant with or after the rate-limiting step for overall folding. The ribozyme forms much of its tertiary structure significantly faster than it reaches this commitment point and the tertiary structure is expected to be stable, suggesting that the commitment to fold along pathways to the native or misfolded states is made from a partially structured intermediate. These results allow the misfolded conformation to be incorporated into a folding framework that reconciles previous data and gives quantitative information about the energetic topology of the folding landscape for this RNA.

Animals↗

PubMed2001

Structure and dimerization of HIV-1 kissing loop aptamers.

Dimerization of two homologous strands of genomic RNA is an essential feature of the retroviral replication cycle. In HIV-1, genomic RNA dimerization is facilitated by a conserved stem-loop structure located near the 5' end of the viral RNA called the dimerization initiation site (DIS). The DIS loop is comprised of nine nucleotides, six of which define an autocomplementary sequence flanked by three conserved purine residues. Base- pairing between the loop sequences of two copies of genomic RNA is necessary for efficient dimerization. We previously used in vitro evolution to investigate a possible structural basis for the marked sequence conservation of the DIS loop. In this study, chemical structure probing, measurements of the apparent dissociation constants, and computer structure analysis of dimerization-competent aptamers were used to analyze the dimers' structure and binding. The selected aptamers were variants of the naturally occurring A and B subtypes. The data suggest that a sheared base-pair closing the loop of the DIS is important for dimerization in both subtypes. On the other hand, the open or closed state of the last base-pair in the stem differed in the two subtypes. This base-pair appeared closed in the subtype A DIS dimer and open in subtype B. Finally, evidence for a cross-talk between nucleotides 2, 5, and 6 was found in some, but not all, loop contexts, indicating some structural plasticity depending on loop sequence. Discriminating between the general rules governing dimer formation and the particular characteristics of individual DIS aptamers helps to explain the affinity and specificity of loop-loop interactions and could provide the basis for development of drugs targeted against the dimerization step during retroviral replication.

Base Pairing↗

PubMed2001

Anatomy of Escherichia coli ribosome binding sites.

During translational initiation in prokaryotes, the 3' end of the 16S rRNA binds to a region just upstream of the initiation codon. The relationship between this Shine-Dalgarno (SD) region and the binding of ribosomes to translation start-points has been well studied, but a unified mathematical connection between the SD, the initiation codon and the spacing between them has been lacking. Using information theory, we constructed a model that treats these three components uniformly by assigning to the SD and the initiation region (IR) conservations in bits of information, and by assigning to the spacing an uncertainty, also in bits. To build the model, we first aligned the SD region by maximizing the information content there. The ease of this process confirmed the existence of the SD pattern within a set of 4122 reviewed and revised Escherichia coli gene starts. This large data set allowed us to show graphically, by sequence logos, that the spacing between the SD and the initiation region affects both the SD site conservation and its pattern. We used the aligned SD, the spacing, and the initiation region to model ribosome binding and to identify gene starts that do not conform to the ribosome binding site model. A total of 569 experimentally proven starts are more conserved (have higher information content) than the full set of revised starts, which probably reflects an experimental bias against the detection of gene products that have inefficient ribosome binding sites. Models were refined cyclically by removing non-conforming weak sites. After this procedure, models derived from either the original or the revised gene start annotation were similar. Therefore, this information theory-based technique provides a method for easily constructing biologically sensible ribosome binding site models. Such models should be useful for refining gene-start predictions of any sequenced bacterial genome.

Base Sequence↗

PubMed2001

A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA.

Viral and plasmid vectors may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes contained in their sequence. Furthermore, replication-defective viral vectors carry the potential risk of recombination with wild-type viruses or activation of oncogenes. A new vector type for minimalistic, immunologically defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal-size gene transfer unit containing the expression cassette, including promoter, gene, and RNA-stabilizing sequence, flanked by two short hairpin oligonucleotide sequences. The resulting vector is a small, linear, covalently closed, dumbbell-shaped molecule. DNA not encoding the desired gene is reduced to a minimum. Here, we transfected colon carcinoma cell lines using cationic lipid, cationic polymer, and electroporation with several MIDGE vectors and corresponding plasmids containing transgenes encoding enhanced green fluorescent protein (eGFP) and human interleukin-2 (hIL-2). Transfection efficiency as measured qualitatively and quantitatively with eGFP was found to be comparable for both vector types. However, hIL-2 secretion and eGFP expression were approximately two- to fourfold higher in most cells transfected with these transgenes using MIDGE vectors compared to the plasmid control. This report demonstrates the advantages of this new vector type and its prospects for ex vivo gene therapy studies.

Cations↗

PubMed2000

High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence.

We investigated the ability of signal peptides of eukaryotic origin (human, mouse, and yeast) to efficiently direct model proteins to the Escherichia coli periplasm. These were compared against a well-characterized prokaryotic signal peptide-OmpA. Surprisingly, eukaryotic signal peptides can work very efficiently in E. coli, but require optimization of codon usage by codon-based mutagenesis of the signal peptide coding region. Analysis of the 5' of periplasmic and cytoplasmic E. coli genes shows some codon usage differences.

Amino Acid Sequence↗

PubMed1997

Identification of cis-acting repressor activity within human immunodeficiency virus type 1 protease sequences.

Human immunodeficiency virus type-1 (HIV-1) Rev overcomes negative elements within viral RNAs to allow expression of gag, pol, and env. The effect of Rev on protein and RNA expression of HIV-1 protease (PR)-containing constructs was investigated utilizing transient transfection of COS cells. Rev, through the Rev response element (RRE), resulted in a large increase in proteolytic activity and cytoplasmic RNA accumulation. Furthermore, Rev increased the level of total RNA produced by a PR-containing construct. The increase in cytoplasmic RNA accumulation in the presence of Rev indicated the presence of cis-acting repressor sequences (CRS) within the RNA produced by this construct. Therefore, components of the construct were analyzed for CRS activity. PR sequences in both sense and antisense orientations exhibited CRS activity. RRE sequences alone conferred a small CRS effect. Additional CRS activity was present within an unspliced RNA containing only nef and LTR sequences. These results indicate a novel form of cis-acting repressor activity within HIV-1 PR; this activity is exerted regardless of the orientation of PR and appears to function at the level of cytoplasmic or nuclear RNA stability.

Animals↗

PubMed2000

The fitness of citrus tristeza virus defective RNAs is affected by the lengths of their 5'- and 3'-termini and by the coding capacity.

Populations of the Closterovirus Citrus tristeza virus (CTV) generally contain defective RNAs (dRNAs) that vary in size, abundance, and sequence. The variation in abundance of the different dRNAs in a population suggests selection for those of higher fitness. To examine factors affecting fitness of dRNAs, we investigated a series of in vitro constructed dRNAs for their ability to be amplified in protoplasts by an efficiently replicated CTV deletion mutant. The minimal sequences required for accumulation of the dRNAs were within the genomic 5' proximal approximately 1 kb and the 3' 270 nucleotides. However, other factors were involved, because a dRNA with only the minimal sequences failed to be replicated. Rescue of a nonviable dRNA by insertion of nonviral sequences between the termini suggested that "spacing" between terminal cis-acting signals influenced fitness. A continuous open reading frame (ORF) through most of the sequences derived from the 5' of the genome was a requirement for dRNA amplification. In general, insertions, deletions, or nucleotide substitutions were tolerated in the dRNAs as long as an ORF was retained, whereas dRNAs with mutations that prematurely terminated the ORF were not viable. To discriminate between a requirement for an essential protein and ribosomal travel, perhaps to present replication signals to the replicase complex, mutations were made to modify the potential protein but still maintain an ORF. Deletions, insertions of nonviral sequences, or switching of reading frames that altered the amino acid sequence of the protein, except the N-terminal 161 amino acids, did not destroy the fitness of the dRNAs. Yet termination of the ORF in the middle of nonviral sequences did destroy the ability of the dRNAs to be amplified. These results suggest that even though a continuous ORF was needed for fitness, its protein product did not affect the amplification of the dRNAs.

Closterovirus↗

PubMed2025

Global Profiling and Analysis of 5' Monophosphorylated mRNA Decay Intermediates.

During RNA turnover, the action of endo- and exo-ribonucleases can yield RNA decay intermediates with specific 5' ends. These RNA decay intermediates have been demonstrated to be the outcome of decapping, microRNA-directed endo-cleavage, or the protected fragments of ribosomes and exon-junction complexes. Therefore, global analysis of RNA decay intermediates can facilitate studies of many RNA decay pathways. In this chapter, we describe a high-throughput sequencing protocol named parallel analysis of RNA ends (PARE), which allows genome-wide profiling of 5' monophosphorylated mRNA decay intermediates from plants or other eukaryotes. Also, we present the tools and scripts necessary for the proper analysis of RNA degradome data obtained from the PARE method. Details and modifications of library construction procedures and bioinformatic analyses to optimize sequencing quality and cope with emerging sequencing platforms and findings are highlighted.

RNA Stability↗

PubMed1992

Dynamical behavior of psb gene transcripts in greening wheat seedlings. I. Time course of accumulation of the pshA through psbN gene transcripts during light-induced greening.

The time course of the accumulation of the transcripts from 13 psb genes encoding a major part of the proteins composing photosystem II during light-induced greening of dark-grown wheat seedlings was examined focusing on early stages of plastid development (0.5 h through 72 h). The 13 genes can be divided into three groups. (1) The psbA gene is transcribed as a single transcript of 1.3 kb in the dark-grown seedlings, but its level increases 5- to 7-fold in response to light due to selective increase in RNA stability as well as in transcription activity. (2) The psbE-F-L-J operon, psbM and psbN genes are transcribed as a single transcript of 1.1 kb, two transcripts of 0.5 and 0.7 kb and a single transcript of 0.3 kb, respectively, in the dark-grown seedlings. The levels of accumulation of every transcript remain unchanged or rather decrease during plastid development under illumination. (3) The psbK-I-D-C gene cluster and psbB-H operon exhibit fairly complicated northern hybridization patterns during the greening process. When a psbC or psbD gene probe was used for northern hybridization, five transcripts differing in length were detected in the etioplasts from 5-day old dark-grown seedlings. After 2 h illumination, two new transcripts of different length appeared. Light induction of new transcripts was also observed in the psbB-H operon.

Blotting, Northern↗

PubMed1996

Dual function of the intron of the rat insulin I gene in regulation of gene expression.

Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

Animals↗

PubMed2000

Rna14p, a component of the yeast nuclear cleavage/polyadenylation factor I, is also localised in mitochondria.

RNA14 was identified as a gene involved in premessenger RNA cleavage and polyadenylation. These processing steps take place in the nucleus, but the Rna14p protein is distributed in both the nucleus and the cytoplasm. By subcellular fractionation, we show here that the cytoplasmic fraction is localised in the mitochondria. In order to understand the role played by Rna14p in mitochondria, we have searched for new thermosensitive alleles of RNA14. We isolated thirteen new mutants. Some of them are deficient in mRNA cleavage and polyadenylation at the restrictive temperature - like the first mutant identified (rna14-1). However, others do not appear to be impaired in any of the steps in RNA metabolism investigated, nor do they appear to be involved in the replication or expression of mitochondrial DNA or in respiration. The localisation data strongly suggest that, besides an essential function in mRNA polyadenylation, the Rna14p protein has a non essential function in mitochondrial metabolism.

Alleles↗

PubMed1999

Cytokine mRNA decay is accelerated by an inhibitor of p38-mitogen-activated protein kinase.

OBJECTIVE: To identify the site(s) in tumor necrosis factor (TNFalpha), interleukin-6 (IL-6), and macrophage inflammatory protein-1alpha (MIP-1alpha) biosynthesis that is blocked by SB202190, a selective inhibitor of p38-mitogen activated protein kinase (p38). MATERIALS: Human blood monocytes isolated by centrifugal elutriation. METHODS: Monocytes were stimulated with lipopolysaccharide in the presence of 0, 0.3, 1 and 3 microM SB202190. Induced TNFalpha, IL-6, and MIP-1alpha protein and mRNA were measured by ELISA and quantitative RT-PCR, respectively. The half-lives of cytokine mRNA levels were determined following treatment of cells with actinomycin D or SB202190. RESULTS: SB202190 suppressed >60% of lipopolysaccharide-induced TNFalpha, IL-6, and MIP-1alpha protein and mRNA expression. Suppressed mRNA levels could be attributed to a >2 to 7-fold reduction in cytokine mRNA half-lives. In contrast, SB202190 did not destabilize mRNAs encoding interferon-induced gene 15 protein and glyceraldehyde-3-phosphate dehydrogenase. CONCLUSIONS: Specific mRNA destabilization represents an important and novel site of action for the cytokine suppressive effects of p38 inhibitors.

Cells, Cultured↗

PubMed2004

Two point mutations identified in emmer wheat generate null Wx-A1 alleles.

In this report, the Wx-A1 mutations carried by a Triticum dicoccoides line from Israel and a Triticum dicoccum line from Yugoslavia are characterized. A single nucleotide insertion in the T. dicoccoides null allele and a single nucleotide deletion in the T. dicoccum null allele each cause frameshift mutations that induce premature termination codons more than 55 nucleotides upstream of the last exon-exon junction. In both mutants, Wx-A1 transcripts were detectable in 10 day post-anthesis endosperm by relative RT-PCR. However, transcript levels of the T. dicoccoides and T. dicoccum null alleles were reduced to approximately 6.5 and 1.5% of wild-type, respectively. Therefore, the lack of Wx-A1 protein in the mutants appears to be largely due to nonsense-mediated mRNA decay. The two mutations described here arose independently, and are not related to either of the Wx-A1 mutations identified in common wheat.

Alleles↗

PubMed2005

The RNA world on ice: a new scenario for the emergence of RNA information.

The RNA world hypothesis refers to a hypothetical era prior to coded peptide synthesis, where RNA was the major structural, genetic, and catalytic agent. Though it is a widely accepted scenario, a number of vexing difficulties remain. In this review we focus on a missing link of the RNA world hypothesis-primitive miniribozymes, in particular ligases, and discuss the role of these molecules in the evolution of RNA size and complexity. We argue that prebiotic conditions associated with freezing, rather than "warm and wet" conditions, could have been of key importance in the early RNA world.

Base Sequence↗

PubMed2005

A role for KEM1 at the START of the cell cycle in Saccharomyces cerevisiae.

KEM1 is a Saccharomyces cerevisiae gene, conserved in all eukaryotes, whose deletion leads to pleiotropic phenotypes. For the most part, these phenotypes are thought to arise from Kem1p's role in RNA turnover, because Kem1p is a major 5'-3' cytoplasmic exonuclease. For example, the exonuclease-dependent role of Kem1p is involved in the exit from mitosis, by degrading the mRNA of the mitotic cyclin CLB2. Here, we describe the identification of a KEM1 truncation, KEM1(1-975), that accelerated the G1 to S transition and initiation of DNA replication when over-expressed. Interestingly, although this truncated Kem1p lacked exonuclease activity, it could efficiently complement another function affected by the loss of KEM1, microtubule-dependent nuclear migration. Taken together, the results we report here suggest that Kem1p might have a previously unrecognized role at the G1 to S transition, but not through its exonuclease activity. Our findings also support the notion that Kem1p is a multifunctional protein with distinct and separable roles.

Cell Cycle Proteins↗

PubMed2000

Comparative sequence analysis of the MECP2-locus in human and mouse reveals new transcribed regions.

Comparative sequence analysis facilitates the identification of evolutionarily conserved regions, that is, gene-regulatory elements, which can not be detected by analyzing one species only. Sequencing of a 152-kb region on human Chromosome (Chr) Xq28 and of the synthenic 123 kb on mouse Chr XC identified the MECP2/Mecp2 locus, which is flanked by the gene coding for Interleukin-1 receptor associated kinase (IRAK/Il1rak) and the red opsin gene (RCP/Rsvp). By comparative sequence analysis, we identified a previously unknown, non-coding 5' exon embedded in a CpG island associated with MECP2/Mecp2. Thus, the MECP2/Mecp2 gene is comprised of four exons instead of three. Furthermore, sequence comparison 3' to the previously reported polyadenylation signal revealed a highly conserved region of 8.5 kb terminating in an alternative polyadenylation signal. Northern blot analysis verified the existence of two main transcripts of 1.9 kb and approximately 10 kb, respectively. Both transcripts exhibit tissue-specific expression patterns and have almost identical short half-lifes. The approximately 10-kb transcript corresponds to a giant 3' UTR contained in the fourth exon of MECP2. The long 3' UTR and the newly identified first intron of MECP2/Mecp2 are highly conserved in human and mouse. Furthermore, the human MECP2 locus is heterogeneous with respect to its DNA composition. We postulate that it represents a boundary between two H3 isochores that has not been observed previously.

3' Untranslated Regions↗

PubMed2026

Esketamine multi-omic biomarker evaluation in major depressive disorder (EMBER-MDD): concept, objectives and methodologies of a non-clinical investigator-initiated study.

Treatment resistance (TR) in major depressive disorder (MDD) affects a substantial minority of patients and is hard to recognize early, delaying intensified care. The Esketamine multi-omic biomarker evaluation in MDD (EMBER-MDD) is a non-interventional, investigator-initiated, in-vitro study within the EU Psych-STRATA programme, analyzing biospecimens collected in the randomized INTENSIFY study and the mirror OBS-TR cohort after participants complete treatment. EMBER-MDD aims to discover individual-omic and integrated multi-omic (hypothesis-free) biomarkers and signatures associated with TR risk, and molecular correlates of clinical response to esketamine nasal spray versus treatment as usual (TAU). Biomaterials will derive from approximately 420 adults with MDD (estimated n = 210 esketamine; n = 210 TAU) and include whole blood, RNA-stabilized whole blood, plasma and serum, sampled at baseline and, when feasible, during and after treatment (up to ~ 5,040 aliquots stored at - 80 °C). Genomics will use baseline DNA genotyping on Illumina Infinium GSA v3.0+MD arrays; epigenomics will profile genome-wide DNA methylation across time points using MethylationEPIC v2.0; transcriptomics will employ mRNA-seq (NovaSeq X/ X Plus); and proteomics/ metabolomics will be generated using high-throughput Olink and/ or Biocrates platforms. Each layer will undergo state-of-the-art preprocessing and analyses (e.g., GWAS/ PRS, EWAS, differential expression, WGCNA, pathway and network analyses), followed by integrative strategies including QTL mapping (meQTL/ eQTL/ pQTL/ mQTL) and intermediate-fusion machine learning with nested cross-validation, explainable AI (SHAP/ LIME) and treatment-effect modelling. All outputs are research-only and will not support individual efficacy, tolerability, or clinical decision-making. The study will deliver robust biosignatures and mechanistic hypotheses to guide future validation and inform stratified, molecularly guided intervention strategies in subsequent prospective trials. Trial registration number: 2023-506617-21-00 and 2025-178-f-S.

Humans↗

PubMed2006

Successful RNA extraction from various human postmortem tissues.

Recently, several authors described the observation that RNA degradation does not correlate with the postmortem interval (PMI), but rather with other parameters like environmental impact and the circumstances of death. Therefore, the question arose if the analysis of gene expression could be a valuable tool in forensic genetics to contribute to the determination of the cause of death. In our study, six human tissues obtained from six individuals with PMI varying between 15 and 118 h were used for total RNA extraction. Quantification was performed using a GAPDH real-time assay, and the quality of mRNA was checked by amplification of different fragment lengths of the GAPDH transcript. In our set of samples, nearly all tissues in all PMI revealed satisfactory results, while skeletal muscle, followed by brain and heart, gave the best results. No correlation between PMI and RNA degradation could be detected, as very good results were observed for all tissues from the individual with the longest PMI. The highly promising results obtained in this study raise hopes that in the near future several fields of forensic investigation may profit from additional information about gene expression patterns and their correlation with pathological findings.

Autopsy↗