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At least 145 records · Page 8Linked to original sources

PubMed2003

Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 x 3 internal loop.

The 5'-leader of HIV-1 RNA controls many viral functions. Nucleocapsid (NC) domains of gag-precursor proteins select genomic RNA for packaging by binding several sites in the leader. One is likely to be a stem defect in SL1 that can adopt either a 1 x 3 internal loop, SL1i (including G247, A271, G272, G273) or a 1 x 1 internal loop (G247 x G273) near a two-base bulge (A269-G270). It is likely that these two conformations are both present and exchange readily. A 23mer RNA construct described here models SL1i and cannot slip into the alternate form. It forms a 1:1 complex with NCp7, which interacts most strongly at G247 and G272 (K(d) = 140 nM). This demonstrates that a linear G-X-G sequence is unnecessary for high-affinity binding. The NMR-based structure shows an easily broken G247:A271 base pair. G247 stacks on both of its immediate neighbors and A271 on its 5'-neighbor; G272 and G273 are partially ordered. A bend in the helix axis between the SL1 stems on either side of the internal loop is probable. An important step in maturation of the virus is the transition from an apical loop-loop interaction to a dimer involving intermolecular interactions along the full length of SL1. A bend in the stem may be important in relieving strain and ensuring that the strands do not become entangled during the transition. A stem defect with special affinity for NCp7 may accelerate the rate of the dimer transformation. This complex could become an important target for anti-HIV drug development, where a drug could exert its action near a high-energy intermediate on the pathway for maturation of the dimer.

Base Sequence↗

PubMed2003

Genomic organization and promoter and transcription regulatory regions for the expression in the anterior brain (sensory vesicle) of Hroth, the otx homologue of the ascidian, Halocynthia roretzi.

Otx (otd in Drosophila) is a well-conserved homeobox gene throughout animal phylogeny and commonly expressed in the anterior part of the embryo. In embryos of the ascidian Halocynthia roretzi, Hroth, the otx homologue in this species, is expressed in the endoderm and the sensory vesicle, the anterior part of the larval ascidian central nervous system (CNS), which has been thought to be homologous to vertebrate forebrain and midbrain. The developmental expression pattern of Hroth is very similar to that of vertebrate counterparts, which leads to a possibility that a similar mechanism may exist in the patterning of the CNS between ascidians and vertebrates. To better understand the mechanism, we decided to undertake analysis of the transcriptional regulatory regions of Hroth. We isolated and determined the nucleotide sequence of the 11.4-kbp region upstream of the translation start site of Hroth. We found that Hroth transcripts are modified likely with spliced leader RNA; therefore, we could not determine the transcription start site. However, first, we identified three introns that are unknown with vertebrate otx genes. Second, we found two regions that are capable of functioning as a promoter through deletion analysis, one of which appeared to be an endogenous promoter of Hroth. We analyzed the 5' upstream region 5402-1473bp, the region between 1473 and 5402 base pairs upstream from the translation start site of Hroth, including the putative endogenous promoter. This region was capable of driving Hroth expression in the sensory vesicle lineage cells as well as some other lineages at the early tail bud stage. Deletion analysis of this region suggested that three regions, 1659-1650bp, 1628-1613bp, and 1542-1473bp are responsible for regulating Hroth expression in the sensory vesicle cells at the tail bud stage. Among these regions, no apparent sequence conservation was observed. The present study has revealed a complex organization of transcription regulatory regions for the ascidian otx.

Amino Acid Sequence↗

PubMed2000

Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo.

The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3'-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247-250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145-157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between -35 and +10 and -95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between -69 and +12, whereas the boundaries of the fully active promoter were between -157 and +54. Computer analysis predicted two stem-loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem-loop structure. Deletion analysis suggested that the upper part of this stem-loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.

Base Sequence↗

PubMed2002

Mobile genetic elements in protozoan parasites.

Mobile genetic elements, by virtue of their ability to move to new chromosomal locations, are considered important in shaping the evolutionary course of the genome. They are widespread in the biological kingdom. Among the protozoan parasites several types of transposable elements are encountered. The largest variety is seen in the trypanosomatids-Trypanosoma brucei, Trypanosoma cruzi and Crithidia fasciculata. They contain elements that insert site-specifically in the spliced-leader RNA genes, and others that are dispersed in a variety of genomic locations. Giardia lamblia contains three families of transposable elements. Two of these are subtleomeric in location while one is chromosome-internal. Entamoeba histolytica has an abundant retrotransposon dispersed in the genome. Nucleotide sequence analysis of all the elements shows that they are all retrotransposons, and, with the exception of one class of elements in T. cruzi, all of them are non-long-terminal-repeat retrotransposons. Although most copies have accumulated mutations, they can potentially encode reverse transcriptase, endonuclease and nucleic-acid-binding activities. Functionally and phylogenetically they do not belong to a single lineage, showing that retrotransposons were acquired early in the evolution of protozoan parasites. Many of the potentially autonomous elements that encode their own transposition functions have nonautonomous counterparts that probably utilize the functions in trans. In this respect these elements are similar to the mammalian LINEs and SINEs (long and short interspersed DNA elements), showing a common theme in the evolution of retrotransposons. So far there is no report of a DNA transposon in any protozoan parasite. The genome projects that are under way for most of these organisms will help understand the evolution and possible function of these genetic elements.

Animals↗

PubMed2002

HIV-1(Lai) genomic RNA: combined used of NMR and molecular dynamics simulation for studying the structure and internal dynamics of a mutated SL1 hairpin.

The genome of all retroviruses consists of two identical copies of an RNA sequence associated in a non-covalent dimer. A region upstream from the splice donor (SL1) comprising a self-complementary sequence is responsible for the initiation of the dimerization. This region is able to dimerize in two conformations: a loop-loop complex or an extended duplex. Here, we solve by 2D NMR techniques the solution structure of a 23-nucleotide sequence corresponding to HIV-1 SL1(Lai) in which the mutation G12-->A12 is included to prevent dimerization. It is shown that this monomer adopts a stem-loop conformation with a seven base pairs stem and a nine nucleotide loop containing the G10 C11 A12 C13 G14 C15 sequence. The stem is well structured in an A-form duplex, while the loop is more flexible even though elements of structure are evident. We show that the structure adopted by the stem can be appreciably different from its relaxed structure when the adenines A8, A9 and A16 in the loop are mechanically constrained. This point could be important for the efficiency of the dimerization. This experimental study is complemented with a 10 ns molecular dynamics simulation in the presence of counterions and explicit water molecules. This simulation brings about information on the flexibility of the loop, such as a hinge motion between the stem and the loop and a labile lattice of hydrogen bonds in the loop. The bases of the nucleotides G10 to C15 were found outside of the loop during a part of the trajectory, which is certainly necessary to initiate the dimerization process of the genuine SL1(Lai) sequence.

Base Sequence↗

PubMed2001

Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA.

The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.

3' Untranslated Regions↗

PubMed2006

Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus.

Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis.

Animals↗

PubMed2004

The 3' termini of small RNAs in Trypanosoma brucei.

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.

Animals↗

PubMed2003

Structure of the intact stem and bulge of HIV-1 Psi-RNA stem-loop SL1.

The Psi-RNA packaging signal of the human immunodeficiency virus type-1 (HIV-1) genome contains a 35 nucleotide stem-loop, termed SL1, which is important for efficient genome packaging during virus assembly and for reverse transcription during infectivity. The predicted secondary structure of SL1 consists of an upper stem with a GC-rich loop that facilitates dimerization, a lower stem, and an intervening bulge (G5, A24-G25-G26) that is both strictly conserved and essential for efficient packaging of the viral genome. The structure of the upper stem in both the kissing and duplex dimer forms have been determined recently. Here, we report the structure of an engineered form of SL1 (SL1(m)) that contains a GAGA tetraloop substituted for the GC-rich loop. This construct does not aggregate and remains monomeric at concentrations up to 1mM, enabling structural studies of the intact stems and bulge. The structure was refined using 1H-13C residual dipolar couplings. The upper stem (C6-G12, C17-G23) is in close agreement with X-ray structures of kissing and duplex dimer forms of related oligoribonucleotides, and nucleotides C1-G4 and C27-G30 form the expected A-helical lower stem. Residues G5 and A24 of the predicted bulge form a G-A mismatch that stacks with the upper stem, and residues G25 and G26 stack between the G-A mismatch and the lower stem in a manner that produces a hole in the center of the bulge and a 25(+/-4) degrees bend between the upper and lower stems. SL1(m) exhibits relatively poor affinity for the HIV-1 nucleocapsid protein, suggesting that the bulge plays other roles in genome packaging.

Base Sequence↗

PubMed2003

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs.

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.

3' Untranslated Regions↗

PubMed2003

Diagnostic genotyping of Old and New World Leishmania species by PCR-RFLP.

We have designed a new genotyping scheme for molecular diagnosis of the different Leishmania species pathogenic to humans. This scheme is based on PCR amplified sequences from the gene for the spliced leader RNA (mini-exon). This target was selected because it is present as tandem repeats (100 to 200 copies) in the genus Leishmania and other kinetoplastida, but is absent from the mammalian hosts and the sandfly vectors. The exon is highly conserved, whereas the intron and non-transcribed spacer region vary in size and sequence among different species. Thus, it was possible to amplify DNA from both Old and New World pathogenic Leishmania complexes using a single pair of primers deriving from the conserved region of the mini-exon tandem repeat. Species identification was performed by digesting mini-exon PCR products with one or two different restriction enzymes. Restriction fragment length polymorphism (RFLP) generated species-specific patterns of bands visualized in agarose gels, which allowed to differentiate each species unequivocally.

Animals↗

PubMed2001

Structure of SL4 RNA from the HIV-1 packaging signal.

The NMR-based structure is described for an RNA model of stem-loop 4 (SL4) from the HIV-1 major packaging domain. The GAGA tetraloop adopts a conformation similar to the classic GNRA form, although there are differences in the details. The type II tandem G.U pairs have a combination of wobble and bifurcated hydrogen bonds where the uracil 2-carbonyl oxygen is hydrogen-bonded to both G,H1 and G,H2. There is the likelihood of a Na(+) ion coordinated to the four carbonyl oxygens in the major groove for these G.U pairs and perhaps to the N7 lone pairs of the G bases as well. A continuous stack of five bases extends over nearly the whole length of the stem to the base of the loop in the RNA 16mer: C15/U14/G13/G5/C6. There is no evidence for a terminal G.A pair; instead, G1 appears quite unrestrained, and A16 stacks on both C15 and G2. Residues G2 through G5 exhibit broadened resonances, especially G3 and U4, suggesting enhanced mobility for the 5'-side of the stem. The structure shows G2/G3/U4 stacking along the same strand, nearly isolated from interaction with the other bases. This is probably an important factor in the signal broadening and apparent mobility of these residues and the low stability of the 16mer hairpin against thermal denaturation.

Base Pairing↗

PubMed2002

Apical loop-internal loop interactions: a new RNA-RNA recognition motif identified through in vitro selection against RNA hairpins of the hepatitis C virus mRNA.

We performed in vitro selection of oligoribonucleotides in order to identify high-affinity motifs recognizing RNA hairpins located at the 3' end (SL1) and at the 5' end (domain IV of the internal ribosome entry site) of the hepatitis C virus mRNA. We selected aptamers constituted by an internal loop complementary to the SL1 apical loop, flanked by G-C-rich double-stranded regions, able to form complexes with a K(d) of 70 nM, at 37 degrees C under ionic conditions close to intracellular ones. The complex involves selective apical loop (SL1)-internal loop (aptamer) interactions. Similar structurally organized aptamers were independently identified against domain IV and were shown to also give rise to such complexes. Apical loop-internal loop interaction could constitute a new recognition motif allowing specific intra- or intermolecular RNA-RNA association.

Base Sequence↗

PubMed2002

Multiple secondary structure rearrangements during HIV-1 RNA dimerization.

HIV-1 RNA dimerization is a complex process that involves a series of RNA refolding events. The monomeric RNA can adopt two alternative conformations that largely determine the efficiency of dimerization. The dimeric RNA also exists in two different conformations, an initial kissing-loop complex and a stable dimer with extended intermolecular base pairing. We describe an ordered RNA folding pathway that incorporates this multitude of HIV-1 RNA conformers. Analysis of mutant transcripts designed to block distinct steps of the refolding cascade supports this model. The folding properties of the wild-type RNA and the defects caused by the mutations can be fully understood in terms of the free energy changes associated with secondary structure rearrangements.

Base Sequence↗

PubMed2002

Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1.

Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.

Amino Acid Sequence↗

PubMed2006

The SL1-SL2 (stem-loop) domain is the primary determinant for stability of the gamma retroviral genomic RNA dimer.

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer.

Base Sequence↗

PubMed1999

Dimer initiation sequence of HIV-1Lai genomic RNA: NMR solution structure of the extended duplex.

The genome of all retrovirus consists of two copies of genomic RNA which are noncovalently linked near their 5' end. A sequence localized immediately upstream from the splice donor site inside the HIV-1 psi-RNA region was identified as the domain responsible for the dimerization initiation. It was shown that a kissing complex and a stable dimer are both involved in the HIV-1Lai RNA dimerization process in vitro. The NCp7 protein activates the dimerization by converting a transient loop-loop complex into a more stable dimer. The structure of this transitory loop-loop complex was recently elucidated by Mujeeb et al. In work presented here, we use NMR spectroscopy to determine the stable extended dimer structure formed from a 23 nucleotides RNA fragment, part of the 35 nucleotides SL1 sequence. By heating to 90 degrees C, then slowly cooling this sequence, we were able to show that an extended dimer is formed. We present evidence for the three dimensional structure of this dimer. NMR data yields evidence for a zipper like motif A8A9.A16 existence. This motif enables the surrounding bases to be positioned more closely and permit the G7 and C17 bases to be paired. This is different to other related sequences where only the kissing complex is observed, we suggest that the zipper like motif AA.A could be an important stabilization factor of the extended duplex.

Dimerization↗

PubMed2002

Translation of Pr55(gag) augments packaging of human immunodeficiency virus type 1 RNA in a cis-acting manner.

The full-length RNA of human immunodeficiency virus type 1 (HIV-1) serves both as a messenger (mRNA) to direct the translation of Pr55(gag) proteins and as genomic or viral particle RNA (vpRNA) to be packaged into virions. In this study, we have assessed a putative cis-acting effect of Pr55(gag) translation on HIV-1 RNA packaging. To pursue this subject, we have measured the relative competence of two distinct types of HIV-1 RNA for being packaged by virus particles under conditions in which only one of them is permissive for production of Pr55(gag). Not surprisingly, wild-type BH10 RNA was packaged at far higher efficiency than that associated with mutant viral RNA that was deleted of RNA packaging signals and incapable of Pr55(gag) production. However, when production of Pr55(gag) was eliminated from the wild-type BH10 viral RNA by insertion of stop codons either in matrix (MA) or in capsid (CA) sequences, regardless of retention of wild-type RNA packaging signals, these Pr55(gag)-deficient viral RNAs were packaged at low levels similar to those observed with viral RNA species that lack RNA packaging signals and are capable of Pr55(gag) generation. Moreover, loss of Pr55(gag) production did not affect stability of the relevant viral RNA; this observation rules out the possibility that lowered packaging efficiency associated with Pr55(gag)-deficient HIV-1 RNA is a result of reduced RNA stability. Taken together, our data demonstrate that cis translation of Pr55(gag) is needed for efficient packaging of HIV-1 RNA.

Animals↗