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PubMed2006

Analysis of a new human parechovirus allows the definition of parechovirus types and the identification of RNA structural domains.

Human parechoviruses (HPeV), members of the Parechovirus genus of Picornaviridae, are frequent pathogens but have been comparatively poorly studied, and little is known of their diversity, evolution, and molecular biology. To increase the amount of information available, we have analyzed 7 HPeV strains isolated in California between 1973 and 1992. We found that, on the basis of VP1 sequences, these fall into two genetic groups, one of which has not been previously observed, bringing the number of known groups to five. While these correlate partly with the three known serotypes, two members of the HPeV2 serotype belong to different genetic groups. In view of the growing importance of molecular techniques in diagnosis, we suggest that genotype is an important criterion for identifying viruses, and we propose that the genetic groups we have defined should be termed human parechovirus types 1 to 5. Complete nucleotide sequence analysis of two of the Californian isolates, representing two types, confirmed the identification of a new genetic group and suggested a role for recombination in parechovirus evolution. It also allowed the identification of a putative HPeV1 cis-acting replication element, which is located in the VP0 coding region, as well as the refinement of previously predicted 5' and 3' untranslated region structures. Thus, the results have significantly improved our understanding of these common pathogens.

Amino Acid Sequence↗
PubMed2005

Effects of donor and acceptor RNA structures on the mechanism of strand transfer by HIV-1 reverse transcriptase.

Template switching during reverse transcription contributes to recombination in human immunodeficiency virus type 1 (HIV-1). Our recent studies suggest that the process can occur through a multi-step mechanism involving RNase H cleavage, acceptor invasion, branch migration, and finally primer terminus transfer. In this study, we analyzed the effects of reverse transcriptase (RT)-pausing, RNase H cleavages and template structure on the transfer process. We designed a series of donor and acceptor template pairs with either minimal pause sites or with pause sites at various locations along the template. Restriction sites within the region of homology allowed efficient mapping of the location of primer terminus transfer. Blocking oligomers were used to probe the acceptor invasion site. Introduction of strong pause sites in the donor increased transfer efficiency. However, the new pauses were not necessarily associated with effective invasion. In this system, the primary invasion occurred at a region of donor cleavage associated with weak pausing. These results together with acceptor structure predictions indicated that a potential invasion site is used only in conjunction with a favorable acceptor structure. Stabilizing acceptor structure at the predicted invasion region lowered the transfer efficiency, supporting this conclusion. Differing from previous studies, terminus transfer occurred at a short distance from the invasion site. Introduction of structure into the acceptor template shifted the location of terminus transfer. Nucleocapsid protein, which can improve cDNA-acceptor interactions, increased transfer efficiency with some shift of terminus transfer closer to the invasion site. Overall results support that the acceptor structure has a major influence on the efficiency and position of the invasion and terminus transfer steps.

Base Sequence↗
PubMed2000

A bidirectional promoter connects the poly(ADP-ribose) polymerase 2 (PARP-2) gene to the gene for RNase P RNA. structure and expression of the mouse PARP-2 gene.

Poly(ADP-ribose) polymerase 2 (PARP-2) is a DNA damage-dependent enzyme that belongs to a growing family of enzymes seemingly involved in genome protection. To gain insight into the physiological role of PARP-2 and to investigate mechanisms of PARP-2 gene regulation, we cloned and characterized the murine PARP-2 gene. The PARP-2 gene consists of 16 exons and 15 introns spanning about 13 kilobase pairs. Interestingly, the PARP-2 gene lies head to head with the gene encoding the mouse RNase P RNA subunit. The distance between the transcription start sites of the PARP-2 and RNase P RNA genes is 114 base pairs. This suggested that regulation of the expression of both genes may be coordinated through a bi-directional promoter. The PARP-2/RNase P RNA gene organization is conserved in the human. To our knowledge, this is the first report of a RNA polymerase II gene and an RNA polymerase III gene sharing the same promoter region and potentially the same transcriptional control elements. Reporter gene constructs showed that the 113-base pair intergenic region was indeed sufficient for the expression of both genes and revealed the importance of both the TATA and the DSE/Oct-1 expression control elements for the PARP-2 gene transcription. The expression of both genes is clearly independently regulated. PARP-2 is expressed only in certain tissues, and RNase P RNA is expressed in all tissues. This suggests that both genes may be subjected to multiple levels of control and may be regulated by different factors in different cellular contexts.

Animals↗
PubMed1984

Alternative splicing of alpha A-crystallin RNA. Structural and quantitative analyses of the mRNAs for the alpha A2- and alpha Ains-crystallin polypeptides.

The eye lens contains a structural protein (alpha-crystallin) composed of two homologous primary gene products, alpha A2 and alpha B2. In certain rodents, there is another alpha-crystallin polypeptide, alpha Ains which is identical with alpha A2 except that it contains an additional peptide between residues 63 and 64 of the alpha A2 chain. The alpha A-crystallin gene encodes the alpha Ains peptide in a separate 69-base pair exon, suggesting that the alpha A2 and alpha Ains mRNAs are derived by alternative RNA splicing. In the present study, we report the isolation of a cloned cDNA (pM alpha AinsCr1) from a cDNA library constructed in the bacterial plasmid pBR322. The nucleotide sequence of pM alpha AinsCr1 in the region of the insert peptide provides compelling evidence that the alpha Ains mRNA is derived from the same gene as the alpha A2 mRNA. S1 nuclease protection experiments, using a DNA fragment from pM alpha AinsCr1, showed that the alternative splicing gives 5 to 10 times more alpha A2 than alpha Ains mRNA. This ratio is comparable to that of the respective polypeptides in the lens. We did not detect an age-related or a differentiation-related difference in the ratio of the alpha A2 to the alpha Ains mRNA or their polypeptides. These results indicate that when the alpha A-crystallin gene is expressed in the lens, it splices the RNA sequences from the insert exon into functional mRNA 10 to 20% of the time.

Aging↗
PubMed1999

Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif.

Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3'-untranslated region of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence data bases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities, and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed by in vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with mitochondrial thioredoxin reductase-2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pilus formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.

Amino Acid Sequence↗
PubMed2003

Small worlds in RNA structures.

I consider conformational spaces of tRNA(phe) defined by sets of suboptimal structures from the perspective of small-world networks. Herein, the influence of modifications on typical small-world network properties and the shape of energy landscapes is discussed. Results indicate that natural modifications influence the degree of local clustering and mean path lengths far more than random or no modifications. High frequencies in the thermodynamic ensemble coincide with high numbers of neighboring structures that one conformation can adopt by one elementary move. Conformation spaces indicate the existence of modular substructures. It can be shown that modifications leave the nature of small-world topology untouched albeit natural modifications have a reasonable enhancing and streamlining effect on the degree of clustering and therefore on the substructures of the conformational space.

Algorithms↗
PubMed1997

Characterization and phylogenetic significance of rhinoceros luteinizing hormone beta (LHbeta) subunit messenger RNA structure, complementary DNA sequence and gene copy number.

The luteinizing hormone (LH) beta subunit gene is expressed in the pituitary glands of all mammals, whereas the closely related chorionic gonadotropin (CG) beta subunit genes have been identified only in primates and equids, and are expressed in placenta. In the case of horses, there is a single-copy equine (e) luteinizing hormone/chorionic gonadotropin hormone beta subunit gene (eLH/CGbeta) that (1) is expressed in both pituitary gland and placenta, (2) encodes a characteristic carboxyl terminal peptide (CTP) extension, and (3) transcribes an atypically elongated 5'-untranslated region (UTR) in both pituitary and placenta. However, it is not known whether similar expression patterns and gene locus characteristics may be exhibited by other members of the order Perissodactyla (equid, rhinoceros and tapir species). To begin to investigate these possibilities, we undertook analysis of the rhinoceros (rn or rhino) LH/(CG?)beta gene locus and the rnLHbeta cDNA. Total RNA isolated from the pituitary gland of a female white rhino was used as template for amplifying rnLHbeta cDNA by reverse transcription-polymerase chain reaction. Following cloning of the amplified cDNA, nucleotide (nt) and deduced amino acid sequences were determined. The first in-frame stop codon occurred at codon position +122, suggesting that the rnLHbeta subunit does not contain a CTP. To assess gene copy number, Southern blot analysis of Indian rhino genomic DNA was performed. The resulting simple hybridization pattern indicated that, as in the horse and donkey, there is a single-copy gene at the rnLH/(CG?)beta gene locus. Primer extension mapping of the pituitary transcriptional start site of the rnLHbeta subunit gene revealed an 8 nt 5'-UTR which is similar to that reported for the majority of mammalian LHbeta transcripts. Northern analysis was consistent with the transcriptional start site findings. We postulate from these data that rhinos diverged from equids prior to the occurrence of the mutations causing CTP expression and adoption of a non-consensus 5'-UTR/proximal promoter region. However, these findings do not rule out the possibility of expression of a placental CGbeta subunit lacking a CTP in rhinos.

Amino Acid Sequence↗
PubMed1994

On the origin of protein synthesis: a speculative model based on hairpin RNA structures.

A speculative model for the origin of protein synthesis based on aminoacylated hairpin RNAs is presented. The model shows how dimerization of these hairpin structures might have triggered the initial synthesis of oligopeptides. The evolution of these dimers seems to lead naturally both to the origin of the tRNA molecule and to the origin of protein synthesis. This co-evolution seems to be confirmed in the determinants of the tRNA identity that might represent the vestiges of the hypothesized model. Finally, the model lends itself to direct experimentation.

Animals↗
PubMedDate not supplied

[Yeast mitochondrial transfer RNA. Structure, coding properties and genome organization].

The up-to-date data on mitochondrial tRNAs of yeast, their structures and peculiarities of these structures, anomalies of the mitochondrial genetic code and anticodons of tRNAs, the structure and number of tRNA genes are reviewed in the present paper. New information concerning 17 types of yeast mitochondrial tRNAs, deciphered by the authors of the paper are given; among them 8 types are first published. The likeness and differences of yeast mitochondrial tRNAs from their cytoplasmic counterparts are discussed by comparison with other organisms.

Base Composition↗
PubMed2006

Functional analyses of RNA structures shared between the internal ribosome entry sites of hepatitis C virus and the picornavirus porcine teschovirus 1 Talfan.

The internal ribosome entry site (IRES) of porcine teschovirus 1 (PTV-1), a member of the Picornaviridae family, is quite distinct from other well-characterized picornavirus IRES elements, but it displays functional similarities to the IRES from hepatitis C virus (HCV), a member of the Flaviviridae family. In particular, a dominant negative mutant form of eIF4A does not inhibit the activity of the PTV-1 IRES. Furthermore, there is a high level (ca. 50%) of identity between the PTV-1 and HCV IRES sequences. A secondary-structure model of the whole PTV-1 IRES has been derived which includes a pseudoknot. Validation of specific features within the model has been achieved by mutagenesis and functional assays. The differences and similarities between the PTV-1 and HCV IRES elements should assist in defining the critical features of this type of IRES.

Animals↗
PubMed1997

Study of RNA structure by pyrene-labeled oligonucleotides.

Fluorescent-labeled oligonucleotides containing 2'-(1-pyrenylmethyl) uridine (Upy) that have G or C adjacent to 3'-site of Upy, I and II, respectively, showed a remarkable increase in the emission intensity when they hybridized with the complementary oligoribonucleotides (ORN). However such change was not observed when they hybridized with the complementary oligodeoxyribonucleotides(ODN). Tm values derived from the UV melting curves for I-ORN and II-ORN duplexes were 5 degrees C and 10 degrees C lower than the normal duplexes without Upy, respectively. The intensity of induced CD based on the pyrene groups for the I-ORN and II-ORN duplexes were a half of that for the corresponding I-ODN and II-ODN duplexes. These results suggest that pyrene groups were released from the stacking interaction upon duplex formation and that the remarkable change of emission intensity was then occurred. Based on these properties, it is suggested that Upy-probes that have G or C adjacent to 3'-site of Upy can be useful to search the single strand region of RNA.

Base Composition↗
PubMed1985

ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors.

Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.

Adenosine Triphosphate↗
PubMed1975

Europium as a fluorescent probe of transfer RNA structure.

The binding of europium(III) to Escherichia coli tRNA-fMet,Glu and to unfractionated E. coli tRNA has been investigated by using the 4-thiouridine sensitization of europium 5-Do yields 7-F1 emission and changes in the lifetime of the 5-Do state of europium reported earlier (J. M. Wolfson and D. R. Kearns (1974), J. Am. Chem. Soc. 96, 3653). Binding of the first 3-4 europium ions is independent and sequential, approximately 600 times stronger than the magnesium binding, and the binding sites are located near the 4-thiouridine residue found at position 8 in a number of E. coli tRNA. Competition experiments suggest the strong binding sites are the same for magnesium and europium. The europium binding properties of both unfractionated E. coli tRNA and purified tRNA-fMet are quite similar, indicating that the location of the strong binding sites and their binding constants are nearly the same for a large group of tRNA. The europium binding properties of native and denatured tRNA are quite different, however.

Binding Sites↗
PubMed1983

Role of RNA structure in splicing: excision of the intervening sequence in yeast tRNA3leu is dependent on the formation of a D stem.

A substitution mutant of the yeast tRNA3leu gene results in the sequence change of GCC to AAA at positions 10, 11, and 12 in the noncoding strand. The ability to form a D stem is lost. Transcription in the heterologous Xenopus germinal vesicle system is not drastically affected, but splicing of the tRNA precursor does not occur. To determine whether this effect is caused by the change in sequence or the change in conformation we constructed two new mutants. In one, mutation results in the sequence change of GGC to TTT at positions 24, 25, and 26. The ability to form a D stem is lost; transcription is unaffected, but excision of the intron does not occur. The other, a double mutant, is characterized by both substitutions described above, and the ability to form a D stem is retained. The precursor derived from the double mutant is accurately spliced in X. laevis germinal vesicle extracts, therefore excision of the intervening sequence appears to depend on the formation of a D stem.

Animals↗
PubMed1980

Transfer RNA structure by carbon NMR: C2 of adenine, uracil and cytosine.

Fourier transform 13C NMR spectra of E. coli tRNA enriched on 13C in either position 2 of adenine (60 atom % 13C) or in position 2 of uracil (82%) and cytosine (63%) were taken at 25.16 MHz over the temperature range 10 degrees - 76 degrees. For C2 of adenine the peak as initially 5 ppm wide, but narrowed to 0.5 ppm as the molecule unfolded. C2 of uracil displayed behavior similar to that of adenine while the cytosine peak, initially relatively narrow at low temperature, sharpened less dramatically. Comparison of spectra at 26.16 MHz and 67.9 MHz showed that the peak widths for folded tRNA were determined largely by chemical shift non-equivalence. T2 T2 measurements suggested that intrinsic line widths of most cytosine C2 peaks were 4 Hz and 2-3 Hz for uracil. Adenine C2 with a directly bonded proton had resonances of about 40 Hz line width. T1 values were measured for C2 of adenine and the ribose carbons of tRNA. Consideration of dipolar relaxation and chemical shift anisotrophy led to a calculated rotational correlation time of 1.6 +/- 0.4 x 10(-8) sec for the adenines and 1.3 +/- 0.3 x 10(-8) sec for the ribose carbons.

Adenine↗
PubMedDate not supplied

Trypanosoma cruzi: RNA structure and post-transcriptional control of tubulin gene expression.

Changes in tubulin expression are among the biochemical and morphological adaptations that occur during the life cycle of Trypanosomatids. To investigate the mechanism responsible for the differential accumulation of tubulin mRNAs in Trypanosoma cruzi, we determine the sequences of alpha- and beta-tubulin transcripts and analyzed their expression during the life cycle of the parasite. Two beta-tubulin mRNAs of 1.9 and 2.3 kb were found to differ mainly by an additional 369 nucleotides at the end of the 3' untranslated region (UTR). Although their transcription rates are similar in epimastigotes and amastigotes, alpha- and beta-tubulin transcripts are 3- to 6-fold more abundant in epimastigotes than in trypomastigotes and amastigotes. Accordingly, the half-lives of alpha- and beta-tubulin mRNAs are significantly higher in epimastigotes than in amastigotes. Transient transfection experiments indicated that positive regulatory elements occur in the 3' UTR plus downstream intergenic region of the alpha-tubulin gene and that both positive and negative elements occur in the equivalent regions of the beta-tubulin gene.

Animals↗
PubMed1987

Partial nucleotide sequence of St. Louis encephalitis virus RNA: structural proteins, NS1, ns2a, and ns2b.

cDNA clones of the St. Louis encephalitis (SLE) virus genome have been obtained and the nucleotide sequence of 4.7 kb corresponding to the 5' terminal half of the genome determined. The genome contains a 5' noncoding region of 98 nucleotides followed by a single continuous open reading frame that encodes three structural proteins in the order capsid (C), membrane precursor (prM)-membrane (M), and envelope (E). Immediately following the C-terminus of E are located nonstructural proteins NS1 through NS3. The SLE amino acid sequence homology with yellow fever (YF), Murray Valley encephalitis (MVE), West Nile (WN), and dengue-2 (DEN) viruses over the sequenced region is 39, 66, 64, and 43%, respectively. The start of each SLE protein has been assigned on the basis of N-terminal sequence data and potential proteolytic cleavage sites homologous with YF and MVE viruses. Flaviviruses have conserved glycosylation sites in prM and NS1 proteins, although only one of the two glycosylation sites in the SLE E protein is conserved in MVE and DEN viruses. An evolutionary tree showing relationships of SLE, MVE, WN, YF, and DEN-2 flaviviruses is proposed on the basis of the amino acid sequences of the C proteins.

Base Sequence↗