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At least 1,117 records · Page 62Linked to original sources
PubMed1997

Characterization of Neurospora mitochondrial group I introns reveals different CYT-18 dependent and independent splicing strategies and an alternative 3' splice site for an intron ORF.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing the N. crassa mitochondrial large rRNA intron by stabilizing the catalytically active structure of the intron core. Here, a comprehensive study of N. crassa mtDNA group I introns identified two additional introns, cob-I2 and the ND1 intron, that are dependent on CYT-18 for splicing in vitro and in vivo. The other seven N. crassa mtDNA group I introns are not CYT-18-dependent and include five that self-splice and two that do not splice under any conditions examined. Some of these introns may require maturases or other proteins for efficient splicing. All but one of the non-CYT-18-dependent introns contain large peripheral extensions of the P5 stem, related to the P5abc structure that blocks CYT-18 binding to the Tetrahymena large rRNA intron. The remaining non-CYT-18-dependent intron, cob-I1, contains a long, peripheral extension of the P9 stem, denoted P9.1, which also impedes CYT-18 binding. Detailed analysis of the CYT-18-dependent ND1 intron showed that two 3' splice sites are used in vitro and in vivo. The proximal, alternative 3' splice site brings the intron open reading frame, which potentially encodes a mobility endonuclease, in frame with the upstream exon, possibly providing a means of expression. Considered together, our results show that group I introns in N. crassa mitochondria use a variety of strategies involving different proteins and/or RNA structures to assist splicing, and they support the hypothesis that CYT-18 and the peripheral RNA structure P5abc are alternative evolutionary adaptations for stabilizing the active structure of the intron core.

Alternative Splicing↗
PubMed1995

Analysis of truncated forms of Bombyx mori glycyl-tRNA synthetase: function of an N-terminal structure in RNA binding.

Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl-tRNA synthetase activity compared to wild type enzyme. Active site titration indicated that this difference in activity was not the result of production of inactive enzyme. Sedimentation and gel filtration experiments indicate that the N-terminally deleted forms and the wild type enzyme were dimers. Deletion of 55 N-terminal residues did not result in significant effects on the Michaelis constants for ATP, glycine, or tRNA, while deletion of 108 N-terminal residues and two internal 64- and 200-residue deletions generated inactive forms. Five forms with C-terminal deletions of 24, 37, 59, 162, and 327 amino acid residues were soluble and intact but lacked detectable pyrophosphate exchange activity or aminoacyl-tRNA synthetase activity. The C-terminal sequence may be required for catalysis or to maintain a stable structure. Zone electrophoresis demonstrated the wild type enzyme bound both tRNA(Gly) and noncognate tRNA(Ala). Deletion of 55 N-terminal residues resulted in altered binding of tRNA(Gly) and eliminated binding of tRNA(Ala). The first 55 N-terminal residues are not essential for catalysis, dimerization, or substrate binding in aminoacylation but are required for RNA binding not associated with aminoacylation.

Adenosine Triphosphate↗
PubMed1984

Blocking of in vitro translation of Paramecium messenger RNAs is due to messenger RNA primary structure.

Paramecium primaurelia mRNAs were translated in vitro in rabbit reticulocyte lysate and the products of translation were analyzed by their size. We show that the large majority of these products are of short but discrete sizes irrespective of the length of the mRNA which directs their synthesis. An illustrative example is given by the translation of mRNA of G surface antigen which directs the synthesis of a 50 kD polypeptide instead of the complete 250 kD protein. Control experiments suggest that the blocking is due to mRNA primary structure.

Animals↗
PubMed1988

CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.

The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop. Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins. Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence. These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins. The stability of CUUCGG hairpins is also utilized in the RNAs of many organisms besides T4.

Base Sequence↗
PubMed2004

In vitro selection and characterization of TCF-1 binding RNA aptamers.

TCF proteins are architectural transcription factors that bind to beta-catenin when the Wnt signaling pathway is activated. TCF-1 is specifically expressed in T-cell lineages and its interaction with beta-catenin is thought to be critical for early T-cell development. However, the mechanisms underlying activation of TCF-1 during T-cell development are not completely understood. With the aim of developing RNA aptamers that bind to TCF-1 and regulate its activity, we screened an RNA library consisting of random sequences of 70 nucleotides and were able to isolate aptamers that bind to TCF-1 with high affinity and specificity. We employed RNase footprinting to characterize the RNA structures and map their binding sites for TCF-1. It is hoped that the selected aptamers will regulate TCF-1 activity in vivo, thereby providing a unique tool for modulating TCF-1 function in early T-cell development.

Animals↗
PubMed1996

Secondary structure model of the coat protein gene of turnip yellow mosaic virus RNA: long, C-rich, single-stranded regions.

The RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of guanosines (15-17%) must impel an RNA structure with a relatively low amount of base pairing and a high incidence of unpaired cytosines. These unpaired cytosines probably function in RNA-protein interactions. To gain Insight into the way the RNA is positioned inside the virion, the secondary structure has been determined of a part of TYMV RNA, including the so-called tymobox, the coat protein gene, and the 3' untranslated region, by structure probing, sequence comparison, and computer predictions. Conservation of secondary structure elements in tymoviruses is not high and does not parallel the conservation of the primary structure. A combination of structure prediction and probing experiments, however, results in a model consisting of structured domains of 100-200 nucleotides interspersed by long unpaired cytosine-rich regions. The latter may interact with the coat protein inside the virion. The structure of some functionally interesting regions of the 3' part of TYMV RNA is also discussed.

Base Sequence↗
PubMed2004

Increased intracellular ionic content is correlated with a decreased perichromatin granule density in old neurons.

The intracellular content of monovalent ions (Na(+), Cl(-), and K(+)) in brain cortical cells from young, adult, and old rats was measured by X-ray microanalysis. By referring the peak of each ion to the respective value of the dry mass, we obtained the intracellular content as a percentage of dry mass. A significant increase of the intracellular content of Na(+), Cl(-), and K(+) was found in old animals. In the same type of cells from rats of different ages, we estimated the density of perichromatin granules (PGs), the RNA structural correlates containing ribonucleoproteins (RNPs). PGs were discovered by Bernhard's method, which allows the preferential staining of RNPs. The PG density (i.e., the number of PGs/microm2 of nuclear area) was significantly lower (-27.8%) in the neurons of old animals. Taken together, the present findings document that in the nuclei of brain cortical cells of old animals the ionic strength is markedly increased and that this alteration may affect chromatin functions (e.g., DNA template activity). The ionic content of the nucleoplasm is reported to modulate the structure of chromatin molecules and regulate gene expression; thus, the increased content of the three ions found by us in old animals, by increasing the viscosity of the nucleoplasm, may affect gene expression by disturbing the inhibition (negative regulation) or potentiation (positive regulation) of RNA polymerase binding. In physiological aging, increased ionic strength may lead to age-related DNA dysfunction as well as to alterations in the processing of RNA structural constituents, such as PGs.

Animals↗
PubMed2004

In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein.

Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids.

Capsid↗
PubMed2000

Genomic organization, proposed alternative splicing mechanisms, and RNA editing structure of GRIK1.

In the present study, we report the genomic reconstruction of the glutamate receptor GluR5 gene (GRIK1, alias GLUR5; 402 kb) by the use of the data available in public databases. This analysis allowed characterization of the exon-intron boundaries, identification of the gene promoter region, presentation of the putative complete sequences of two GluR5 mRNA isoforms, and definition of the structure of GluR5 pre-mRNA surrounding the Q/R editing site.

Alternative Splicing↗
PubMed2007

Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission.

The 3' untranslated regions (3'UTRs) of flaviviruses are reviewed and analyzed in relation to short sequences conserved as direct repeats (DRs). Previously, alignments of the 3'UTRs have been constructed for three of the four recognized flavivirus groups, namely mosquito-borne, tick-borne, and nonclassified flaviviruses (MBFV, TBFV, and NCFV, respectively). This revealed (1) six long repeat sequences (LRSs) in the 3'UTR and open-reading frame (ORF) of the TBFV, (2) duplication of the 3'UTR of the NCFV by intramolecular recombination, and (3) the possibility of a common origin for all DRs within the MBFV. We have now extended this analysis and review it in the context of all previous published analyses. This has been achieved by constructing a robust alignment between all flaviviruses using the published DRs and secondary RNA structures as "anchors" to reveal additional homologies along the 3'UTR. This approach identified nucleotide regions within the MBFV, NKV (no-known vector viruses), and NCFV 3'UTRs that are homologous to different LRSs in the TBFV 3'UTR and ORF. The analysis revealed that some of the DRs and secondary RNA structures described individually within each flavivirus group share common evolutionary origins. The 3'UTR of flaviviruses, and possibly the ORF, therefore probably evolved through multiple duplication of an RNA domain, homologous to the LRS previously identified only in the TBFV. The short DRs in all virus groups appear to represent the evolutionary remnants of these domains rather than resulting from new duplications. The relevance of these flavivirus DRs to evolution, diversity, 3'UTR enhancer function, and virus transmission is reviewed.

3' Untranslated Regions↗
PubMed1980

Chemical probes for higher-order structure in RNA.

Three chemical reactions can probe the secondary and tertiary interactions of RNA molecules in solution. Dimethyl sulfate monitors the N-7 of guanosines and senses tertiary interactions there, diethyl pyrocarbonate detects stacking of adenosines, and an alternate dimethyl sulfate reaction examines the N-3 of cytidines and thus probes base pairing. The reactions work between 0 degrees C and 90 degrees C and at pH 4.5--8.5 in a variety of buffers. As an example we follow the progressive denaturation of yeast tRNAPhe terminally labeled with 32P as the tertiary and secondary structures sequentially melt out. A single autoradiograph of a terminally labeled molecule locates regions of higher-order structure and identifies the bases involved.

Alkylating Agents↗
PubMed1993

Automatic display of RNA secondary structures.

A set of programs written in C language with the GL library and under UNIX has been developed for generating compact, pleasant and non-overlapping displays of secondary structures of ribonucleic acids. The first program, rnasearch, implements a new search procedure that dynamically rearranges overlapping portions of the two-dimensional drawing while preserving clear and readable displays of the two-dimensional structure. The algorithm is fast (the execution time for the command rnasearch is 38.6 s for the 16S rRNA of Escherichia coli with 1542 bases), accepts outputs from two-dimensional prediction programs and therefore allows for rapid comparison between the various two-dimensional folds generated. A second program, rnadisplay, allows the graphical display of the computed two-dimensional structures on a graphics workstation. Otherwise, it is possible to obtain a paper output of the two-dimensional structure by using the program print2D which builds a Postscript file. Moreover the two-dimensional drawing can be labelled for representing data coming from chemical modifications and/or enzymatic cleavages. Application to a few secondary structures such as RNaseP, 5S rRNA and 16S rRNA are given.

Algorithms↗
PubMed2004

Recognition of duplex RNA by helix-threading peptides.

Many important biological processes, from the interferon antiviral response to the generation of microRNA regulators of translation, involve duplex RNA. Small molecules capable of binding duplex RNA structures with high affinity and selectivity will be useful in regulating these processes and, as such, are valuable research tools and potentially therapeutic. In this paper, the synthesis and duplex RNA-binding properties of EDTA.Fe-modified peptide-intercalator conjugates (PICs) are described. Peptide appendages at the 4- and 9-positions of the planar acridine ring system render these PICs threading intercalators, directing the substituents into both grooves of double helical RNA simultaneously. Directed hydroxyl radical cleavage experiments conducted with varying RNA stem-loop structures indicate a preferred binding polarity with the N- and C-termini of the PIC in the minor and major grooves, respectively. However, this binding polarity is shown to be dependent on both the structure of the PIC and the RNA secondary structure adjacent to the intercalation site. Definition of the minimal RNA structure required for binding to one of these PICs led to the identification of an intercalation site in a pre-microRNA from Caenorhabditis elegans. Results presented will guide both rational design and combinatorial approaches for the generation of new RNA binding PICs and will continue to facilitate the identification of naturally occurring RNA targets for these small molecules.

Edetic Acid↗
PubMed1993

Expression of bacteriophage T4 gene 25 is regulated via RNA secondary structure in the translational initiation region.

Analysis of the nucleotide sequence in the 5' flanking region of bacteriophage T4 gene 25 revealed three potential Shine and Dalgarno sequences, SD1, SD2 and SD3, with a spacing of 8, 17 and 27 nucleotides from the initiation codon of this gene, respectively. Results of our experiments in the bacteriophage T7 expression system clearly demonstrate that the SD3 sequence is required for efficient expression of gene 25. We propose the existence of a stem-loop structure that includes SD1 and SD2 sequences and brings the SD3 sequence to a favourable spacing with the initiation codon of gene 25. Since the predicted secondary structure in the translational initiation region of gene 25 is relatively unstable and the SD3 sequence, GAGG, is more typical than the SD1 sequence, GAG, we suggest that this structure could control the level of gene expression.

Bacteriophage T4↗