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At least 811 records · Page 45Linked to original sources
PubMed1988

Nucleotide sequence of RNA segment 7 and the predicted amino sequence of M1 and M2 proteins of FPV/Weybridge (H7N7) and WSN (H1N1) influenza viruses.

Since the gene products (M1 and M2) of influenza virus RNA segment 7 have been implicated in host range restriction, sensitivity to the drug amantadine, virus yield in chicken embryos as well as in virus assembly and morphology, we have determined the nucleotide sequence of this RNA segment for an avian [A/FPV/Weybridge (H7N7)] and a human [A/WSN/33 (H1N1)] virus and compared it to that of the other influenza A virus strains. The results show that all ten strains of influenza A virus contain an identical number of nucleotides (1027 bases) in RNA segment 7 and an identical number of amino acids in M1 (252 aa) and M2 (97 aa) proteins. The observed amino acid changes are conservative in nature suggesting the requirement of a critical structure of both proteins in virus assembly. Furthermore, the presence of some consistent amino acid substitutions among different human and avian strains also supports the possible existence of host range and drug resistance determinants in M1 and M2 proteins.

Amino Acid Sequence↗
PubMed2006

Kinetics of RNA refolding in dynamic equilibrium by 1H-detected 15N exchange NMR spectroscopy.

By implementing new NMR methods that were designed to map very slow exchange processes we have investigated and characterized the refolding kinetics of a thermodynamically stable 34mer RNA sequence in dynamic equilibrium. The RNA sequence was designed to undergo a topologically favored conformational exchange between different hairpin folds, serving as a model to estimate the minimal time required for more complex RNA folding processes. Chemically prepared RNA sequences with sequence-selective (15)N labels provided the required signal separation and allowed a straightforward signal assignment of the imino protons by HNN correlation experiments. The 2D version of the new (1)H-detected (15)N exchange spectroscopy (EXSY) pulse sequence provided cross-peaks for resonances belonging to different folds that interchange on the time scale of longitudinal relaxation of (15)N nuclei bound to imino protons. The 34mer RNA sequence exhibits two folds which exchange on the observable time scale (tau(obs) approximately T(1){(15)Nu} < 5 s) and a third fold which is static on this time scale. A 1D version of the (15)N exchange experiment allowed the measurement of the exchange rates between the two exchanging folds as a function of temperature and the determination of the corresponding activation energies E(a) and frequency factors A. We found that the refolding rates are strongly affected by an entropically favorable preorientation of the replacing strand. The activation energies are comparable to values obtained for the slow refolding of RNA sequences of similar thermodynamic stability but less favorable topology.

Base Sequence↗
PubMed2002

Functional analysis of the murine sarcoma virus RNA packaging sequence.

We investigated the features of the Moloney murine sarcoma virus leader sequence necessary for RNA packaging function by using a deletion analysis approach. We found that sequences that extend beyond those characterized genetically in previous reports are important for optimal packaging efficiency. A fragment covering a minimum of four potential stem-loop structures is required for the shortest packaging element compatible with gene transfer. Our results reveal the extent to which each of the segments of the packaging sequence contribute to packaging efficiency.

5' Untranslated Regions↗
PubMed2006

Sequence dependence of the stability of RNA hairpin molecules with six nucleotide loops.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the types GCGXUAAUYCGC and GGUXUAAUYACC with Watson-Crick loop closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.1 to 5.1 kcal/mol. These results agree with the model previously developed [Vecenie, C. J., and Serra, M. J. (2004) Biochemistry 43, 11813] to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on the 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Thermodynamic parameters are also reported for hairpin formation in 1 M NaCl by RNA sequence of the types GACGXUAAUYUGUC and GGUXUAAUYGCC with GU base pair closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.6 to 5.3 kcal/mol. These results allow the development of a model for predicting the stability of hairpin loops closed by GU base pairs. DeltaG degrees (37L(n) (kcal/mol) = DeltaG degrees (37i(n) - 0.8 (if the first mismatch is GA) - 0.8 (if the first mismatch is GG and the loop is closed on the 5' side by a purine). Note that for these hairpins, the stability of the loops does not depend on DeltaG degrees (37MM). For hairpin loops closed by GU base pairs, the DeltaG degrees (37i(n) values, when n = 4, 5, 6, 7, and 8, are 4.9, 5.0, 4.6, 5.0, and 4.8 kcal/mol, respectively. The model gives good agreement when tested against six naturally occurring hairpin sequences. Thermodynamic values for terminal mismatches adjacent to GC, GU, and UG base pairs are also reported.

Base Composition↗
PubMed1988

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis.

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.

Base Sequence↗
PubMed1970

Terminal-sequence studies of high-molecular-weight ribonucleic acid. The 3'-termini of rabbit reticulocyte ribosomal RNA.

Sequences of the polynucleotide chains of RNA found in the large and small ribosomal subunits of rabbit reticulocytes have been determined from the 3'-end by use of periodate oxidation and condensation with [(3)H]isoniazid and by stepwise degradation. By these methods the hexanucleotide sequences have been found as -pGpUpUpUpGpU for the 28S RNA and -pGpUpCpGpCpU for the 6S RNA of the large ribosomal subunit and the octanucleotide sequence -pGpApUpCpApUpUpA for the 18S rRNA of the small ribosomal subunit. These sequences are present in at least 70% of all the RNA molecules and are discussed in relation to the specific cleavage of rRNA from its precursors and the role of multiple cistrons for rRNA in the DNA of higher organisms. The feasibility of using the method for longer sequence determinations is discussed.

Adenine Nucleotides↗
PubMed1984

The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing.

3' Processing of sea urchin H3 histone pre-mRNA depends on a small nuclear RNP which contains an RNA of nominally 60 nucleotide length, referred to below as U7 RNA. The U7 RNA can be enriched by precipitation of sea urchin U-snRNPs with human systematic lupus erythematosus antiserum of the Sm serotype. We have prepared cDNA clones of U7 RNA and determined by hybridization techniques that this RNA is present in sea urchin eggs at 30-fold lower molar concentration than U1 RNA. The RNA sequences derived from an analysis of eight U7 cDNA clones show neither homologies nor complementarities to any other know U-RNAs. The 3' portion of the presumptive RNA sequence can be folded into a stem-loop structure. The 5'-terminal sequences would be largely unstructured as free RNA. Their most striking feature is their base complementarity to the 3' conserved sequences of histone pre-mRNAs. Six out of nine bases of the conserved CAAGAAAGA sequence of the histone mRNA precursor and 13 out of 16 nucleotides from the conserved palindrome can be base paired with presumptive U7 RNA sequence, suggesting a unique hybrid structure for a processing intermediate formed from histone precursor and U7 RNA.

Animals↗
PubMed1996

A one-hybrid system for detecting RNA-protein interactions.

BACKGROUND: mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs. Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNA-protein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences. The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals. The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA-protein interactions. RESULTS: Our results show that fusion of the RNA binding protein, R17, to N causes antitermination in a beta-galactosidase reporter construct that has the R17 binding site substituted for the normal N target sequence. This system can both detect low affinity interactions as well as discriminate between binding events with equilibrium dissociation constants from 10(-5) to 10(-8)M. The differences in antitermination activity with various mutant binding sites can be reliably detected by colony colour on X-Gal plates as well as by liquid culture assay. CONCLUSIONS: We have demonstrated that N protein can cause antitermination through a heterologous RNA-protein interaction and that the system is capable of detecting RNA-protein interactions of differing affinities. This approach may also be useful in screening libraries for proteins that bind to novel RNA regulatory elements. Our results are also consistent with a model of N protein function in which binding to the nascent transcript increases the effective concentration of N in the vicinity of RNA polymerase leading to antitermination.

Capsid↗
PubMed2003

Specific RNA interference in psbP genes encoded by a multigene family in Nicotiana tabacum with a short 3'-untranslated sequence.

RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3'-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3'-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3'-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3'-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.

3' Untranslated Regions↗
PubMed1989

A second trans-spliced RNA leader sequence in the nematode Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, the 22-nucleotide RNA sequence called the spliced leader (SL) is trans-spliced from the 100-nucleotide-long SL RNA to some mRNAs. We have identified a trans-spliced leader (SL2) whose sequence differs from that of the original spliced leader (SL1), although both are 22 nucleotides long. By primer-extension sequencing, SL2 but not SL1 was shown to be present at the 5' end of the mRNA encoded by one of the four glyceraldehyde-3-phosphate dehydrogenase genes. The other three glyceraldehyde-3-phosphate dehydrogenase genes encode mRNAs that have the SL1 but not the SL2 sequence at their 5' ends. Therefore, the trans-splicing process can discriminate the transfer of SL1 from that of SL2 in a gene-specific manner.

Animals↗
PubMed1999

Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis.

Y RNAs are small 'cytoplasmic' RNAs which are components of the Ro ribonucleoprotein (RNP) complex. The core of this complex, which is found in the cell nuclei of higher eukaryotes as well as the cytoplasm, is composed of a complex between the 60 kDa Ro protein and Y RNAs. Human cells contain four distinct Y RNAs (Y1, Y3, Y4 and Y5), while other eukaryotes contain a variable number of Y RNA homologues. When detected in a particular species, the Ro RNP has been present in every cell type within that particular organism. This characteristic, along with its high conservation among vertebrates, suggests an important function for Ro RNP in cellular metabolism; however, this function has not yet been definitively elucidated. In order to identify conserved features of Y RNA sequences and structures which may be directly involved in Ro RNP function, a phylogenetic comparative analysis of Y RNAs has been performed. Sequences of Y RNA homologues from five vertebrate species have been obtained and, together with previously published Y RNA sequences, used to predict Y RNA secondary structures. A novel RNA secondary structure comparison algorithm, the suboptimal RNA analysis program, has been developed and used in conjunction with available algorithms to find phylogenetically conserved secondary structure models for YI, Y3 and Y4 RNAs. Short, conserved sequences within the Y RNAs have been identified and are invariant among vertebrates, consistent with a direct role for Y RNAs in Ro function. A subset of these are located wholly or partially in looped regions in the Y3 and Y4 RNA predicted model structures, in accord with the possibility that these Y RNAs base pair with other cellular nucleic acids or are sites of interaction between the Ro RNP and other macromolecules.

Animals↗
PubMed1996

The S RNA genomic sequences of Inkoo, San Angelo, Serra do Navio, South River and Tahyna bunyaviruses.

The complete nucleotide sequences of the small (S) genomic RNA segments of five California (CAL) serogroup bunyaviruses (two Inkoo virus strains, San Angelo virus, Serra do Navio virus, South River virus and Tahyna virus) were determined. In agreement with previously published data concerning CAL serogroup viruses, the nucleocapsid (N) and non-structural (NSs) proteins were encoded in over-lapping open reading frames (ORFs). All N protein ORFs were 708 nucleotides in length and encoded a 235 amino-acid gene product. The NSs ORFs were either 279 or 294 nucleotides in length, which would encode 92 or 97 amino-acid proteins, respectively. Comparative analysis of the nucleotide sequences and amino acids corresponding to the nucleocapsid protein resulted in a predicted relationship among these viruses that generally agreed with those determined by serology. The only exception was Inkoo virus, where comparisons based on the S RNA sequence and partial M RNA sequence suggest that this virus is more similar to Jamestown Canyon virus of the Melao complex than it is to viruses such as Tahyna and La Crosse viruses of the California encephalitis complex.

Amino Acid Sequence↗
PubMed1996

The nucleotide sequence of RNA-1 of Indian peanut clump furovius.

The nucleotide sequence of RNA-1 of an isolate of the H serotype of Indian peanut clump virus (IPCV-H) was shown to comprise 5,841 nucleotides. The RNA contains three open reading frames (ORF) which are between nucleotides 133 and 3,522, nucleotides 3,526 and 5,103 (assuming expression by suppression of the ORF 1 termination codon) and nucleotides 5,168 and 5,539. The encoded polypeptides have M(r), of 129,687 (p130), 60,188 (p60) and 14,281 (p14). ORF 2 is thought to be expressed by suppression of the termination codon of ORF 1 to produce a M(r) 189,975 product (p190). p130 contains sequences characteristic of proteins with methyl transferase and NTP-binding properties and p190 contains these and sequences characteristic of RNA-dependent RNA polymerases. The nucleotide sequence of IPCV RNA-1 is similar to that of peanut clump virus (PCV) and corresponding encoded polypeptides are 88% (p130), 95% p60 and 75% (p14) identical. The sequences of the translation products are also similar to those of soil-borne wheat mosaic virus and barley stripe mosaic virus. Oligonucleotide primers, designed on the basis of the sequences of RNA-1 of IPCV and PCV, were effective in reverse transcription-PCR amplification of these RNAs and that of IPCV isolates of the serologically distinct L and T serotypes.

Amino Acid Sequence↗
PubMedDate not supplied

Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate.

The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.

Base Sequence↗
PubMed2004

On the stability of different experimental dimeric structures of the SL1 sequence from the genomic RNA of HIV-1 in solution: a molecular dynamics simulation and electrophoresis study.

SL1 is a stem-loop RNA sequence from the genome of HIV-1 thought to be the initiation site for the dimerization of the retroviral genomic RNA. The aim of this study is to check the stability in solution of different experimental dimeric structures available in the literature. Two kinds of dimer have been evidenced: an extended duplex looking like a double helix with two internal bulges and a kissing complex in which the monomers with a stem/loop conformation are linked by intermolecular loop-loop interactions. Two divergent experimental structures of the kissing complex from the Lai isolate are reported in the literature, one obtained from NMR (Mujeeb et al., Nature Structural Biology, 1998, Vol. 5, pp. 432-436) and the other one from x-ray crystallography (Ennifar et al., Nature Structural Biology, 2001, Vol. 8, pp. 1064-1068). A crystallographic structure of the Mal isolate was also reported (Ennifar et al., Nature Structure Biology, 2001, Vol. 8, pp. 1064-1068). Concerning the extended duplex, a NMR structure is available for Lai (Girard et al., Journal of Biomolecular Structure and Dynamics, 1999, Vol. 16, pp. 1145-1157) and a crystallographic structure for Mal (Ennifar et al., Structure, 1999, Vol. 7, pp. 1439-1449). Using a molecular dynamics technique, all these experimental structures have been simulated in solution with explicit water and counterions. We show that both extended duplex structures are stable. On the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment. Finally, the NMR structure of the Lai loop-loop kissing complex remains globally stable over a 20 ns MD simulation, although large rearrangements occur at the level of the stem/loop junctions that are flexible, as shown from free energy calculations. These results are compared to electrophoresis experiments on dimer formation.

Computer Simulation↗
PubMed1992

Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1.

Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.

Amino Acid Sequence↗
PubMed1990

AUUUA motifs are dispensable for rapid degradation of the mouse c-myc RNA.

Sequence determinants responsible for c-myc RNA rapid turn-over are localized within the 3' non-coding region which is mainly characterized by the presence of two polyadenylation signals and a high content in A and U. Although the AUUUA/UUAUUUA motif is commonly thought to specify a whole class of unstable RNAs coding for various onco-proteins and cytokines, site-directed mutagenesis showed that both of the two such sequences found in the mouse c-myc RNA are dispensable for rapid RNA degradation. Although less efficient than the whole 3' non-coding region, the last 50 nucleotides of c-myc RNA, mainly made up of U and A and devoid of AUUUA/UUAUUUA motif, are sufficient to confer instability to the coding sequence.

Animals↗
PubMed2023

Genome-wide identification of transcriptional enhancers during human placental development and association with function, differentiation, and disease&#x2020;.

The placenta is a dynamic organ that must perform a remarkable variety of functions during its relatively short existence in order to support a developing fetus. These functions include nutrient delivery, gas exchange, waste removal, hormone production, and immune barrier protection. Proper placenta development and function are critical for healthy pregnancy outcomes, but the underlying genomic regulatory events that control this process remain largely unknown. We hypothesized that mapping sites of transcriptional enhancer activity and associated changes in gene expression across gestation in human placenta tissue would identify genomic loci and predicted transcription factor activity related to critical placenta functions. We used a suite of genomic assays [i.e., RNA-sequencing (RNA-seq), Precision run-on-sequencing (PRO-seq), and Chromatin immunoprecipitation-sequencing (ChIP-seq)] and computational pipelines to identify a set of >20&#xa0;000 enhancers that are active at various time points in gestation. Changes in the activity of these enhancers correlate with changes in gene expression. In addition, some of these enhancers encode risk for adverse pregnancy outcomes. We further show that integrating enhancer activity, transcription factor motif analysis, and transcription factor expression can identify distinct sets of transcription factors predicted to be more active either in early pregnancy or at term. Knockdown of selected identified transcription factors in a trophoblast stem cell culture model altered the expression of key placental marker genes. These observations provide a framework for future mechanistic studies of individual enhancer-transcription factor-target gene interactions and have the potential to inform genetic risk prediction for adverse pregnancy outcomes.

Humans↗