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At least 235 records · Page 13Linked to original sources
PubMed2002

RNA structure and packaging signals in the 5' leader region of the human immunodeficiency virus type 1 genome.

The leader region of the human immunodeficiency virus type 1 (HIV-1) genome has a highly folded structure, comprising at least two RNA stem-loops [the transactivation response (TAR) and poly(A) hairpins] near its 5' end and four others (SL1 to SL4) downstream. Each of these stem-loops contributes to the function of the HIV-1 packaging signal, which efficiently targets genomic RNA into nascent virions. The central 140-base region of the leader, which includes the U5 and primer binding site (PBS) sequences, is also believed to adopt a complex structure, but the nature of this structure and its possible role in RNA packaging have not been extensively explored. Here we report a mutational analysis identifying at least three separate loci within the U5-PBS region which, when mutated, impair both HIV-1 packaging specificity and infectivity in a single-round proviral assay. In common with those of all previously described packaging signals in the leader, the function of one of these loci appeared to depend on secondary structure rather than on sequence alone. By contrast, the activity of the other two loci did not correlate with any predicted conformations. Moreover, unlike SL1 to SL4, the TAR, poly(A), and U5-PBS hairpins were not bound with high affinity by the nucleocapsid portion of the HIV-1 Gag protein in vitro, implying that they contribute to packaging through a mechanism distinct from that of SL1 to SL4. Our findings confirm the existence and importance of secondary structure around the PBS and demonstrate that functional packaging signals are distributed across the entire HIV-1 leader.

5' Untranslated Regions↗
PubMed1984

Ribosomal RNA structure in the diploid and phylogenetically polyploid amphibian species Hyla and Odontophrynus.

Ribosomal RNA of the diploid amphibian species Hyla chrysoscelis and Odontophrynus americanus is structurally modified by hidden breaks. Phylogenetically polyploid related species like the tetraploid Hyla versicolor, the tetraploid Odontophrynus americanus and the octoploid Ceratophrys ornata do not show hidden breaks in ribosomal RNA. Structural modifications of rRNA molecules in diploid amphibians has no detectable effect on the ribosomal activity in vitro.

Amphibians↗
PubMed1989

An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus.

The RNA genome of the hepatitis delta virus (HDV) appears to be made up of two parts: a small domain with a high degree of sequence conservation and structural features likely to promote replication; plus a second, larger domain that is less conserved and encodes the delta antigen. This report focuses on one of the several sets of data that have led to the proposal of this model: the existence of a novel structural element in HDV genomic RNA. This structural element lies within the highly conserved domain of HDV RNA and may be related to the local tertiary structure previously mapped to the central conserved region of the plant viroid genome. Both elements occur in regions with no apparent coding capacity and are distinctively responsive to ultraviolet (UV) light. Transcripts containing partial and full-length genomic sequences of HDV readily undergo a UV-induced crosslinking reaction, which establishes a covalent bond between two noncontiguous segments. By locking two segments of the overall structure into place, this crosslink has permitted the unbranched, rodlike model of HDV RNA to be examined and confirmed in the portion of the RNA analyzed. The clustering of the novel tertiary structure and the recently discovered self-cleavage sites into a highly conserved, but apparently noncoding, portion of the genome defines a viroid-like domain in HDV RNA and raises questions about the possible events leading up to the association of free-living RNAs with messenger RNAs and other RNA molecules.

DNA↗
PubMed1999

Genetic analysis of a unique human immunodeficiency virus type 1 (HIV-1) with a primer binding site complementary to tRNAMet supports a role for U5-PBS stem-loop RNA structures in initiation of HIV-1 reverse transcription.

Human immunodeficiency virus type 1 (HIV-1) exclusively uses tRNA3Lys to initiate reverse transcription. A novel HIV-1 mutant which stably utilizes tRNAMet rather than tRNA3Lys as a primer was previously identified [HXB2(Met-AC] (S.-M. Kang, Z. Zhang, and C. D. Morrow, J. Virol. 71:207-217, 1997). Comparison of RNA secondary structures of the unique sequence (U5)-primer binding site (PBS) viral RNA genome alone or complexed with tRNAMet of HXB2(Met-AC) revealed structural motifs in common with the U5-PBS of the wild-type virus. In the current study, mutations were constructed to alter the U5-PBS structure and disrupt the U5-PBS-tRNAMet interaction of the virus derived from HXB2(Met-AC). All of the mutant viruses were infectious following transfection and coculture with SupT1 cells. Analysis of the initiation of reverse transcription revealed that some of the mutants were impaired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral constructs, one predicted to completely disrupt the stem-loop structure in U5 and the other predicted to destabilize contact regions of U5 with tRNAMet, reverted back to contain a PBS complementary to tRNA3Lys. All other mutants maintained a PBS complementary to tRNAMet after in vitro culture, although all contained multiple nucleotide substitutions within the U5-PBS from the starting proviral clones. Most interestingly, a viral mutant containing a 32-nucleotide deletion between nucleotides 142 and 173, encompassing regions in U5 which interact with tRNAMet, maintained a PBS complementary to tRNAMet following in vitro culture. All of the proviral clones recovered from this mutant, however, contained an additional 19-nucleotide insertion in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structures similar to those modeled from HXB2(Met-AC). These results provide strong genetic evidence that multiple sequence and structural elements in U5 in addition to the PBS are involved in the interaction with the tRNA used for initiation of reverse transcription.

Base Sequence↗
PubMed2005

A highly conserved 6S RNA structure is required for regulation of transcription.

6S RNA, a highly abundant noncoding RNA, regulates transcription through interaction with RNA polymerase in Escherichia coli. Computer searches identified 6S RNAs widely among gamma-proteobacteria. Biochemical approaches were required to identify more divergent 6S RNAs. Two Bacillus subtilis RNAs were found to interact with the housekeeping form of RNA polymerase, thereby establishing them as 6S RNAs. A third B. subtilis RNA was discovered with distinct RNA polymerase-binding activity. Phylogenetic comparison and analysis of mutant RNAs revealed that a conserved secondary structure containing a single-stranded central bulge within a highly double-stranded molecule was essential for 6S RNA function in vivo and in vitro. Reconstitution experiments established the marked specificity of 6S RNA interactions for sigma(70)-RNA polymerase, as well as the ability of 6S RNA to directly inhibit transcription. These data highlight the critical importance of structural characteristics for 6S RNA activity.

Bacillus subtilis↗
PubMed1994

An RNA-binding peptide from bovine immunodeficiency virus Tat protein recognizes an unusual RNA structure.

The human immunodeficiency virus (HIV) Tat protein binds specifically to an RNA hairpin, TAR, located at the 5' end of its mRNA. Tat uses a single arginine residue within a short region of basic amino acids to recognize a bulge region in TAR. Here we show that a 17 amino acid arginine-rich peptide from the bovine immunodeficiency virus (BIV) Tat protein also binds to an RNA hairpin at the 5' end of its mRNA (BIV TAR), but recognizes different structural features of the RNA. Mutagenesis, RNase mapping, and chemical interference experiments indicate that bulge and stem regions of BIV TAR are recognized simultaneously by the BIV peptide and that the RNA adopts an unusual structure. BIV Tat binds to its TAR site with high affinity and specificity and, unlike HIV Tat, does not appear to use cellular proteins to stabilize RNA binding in vivo. Thus, two related viral activators have evolved rather distinct ways to recognize their RNA targets.

Amino Acid Sequence↗
PubMed2000

Phylogenetically enhanced statistical tools for RNA structure prediction.

MOTIVATION: Methods that predict the structure of molecules by looking for statistical correlation have been quite effective. Unfortunately, these methods often disregard phylogenetic information in the sequences they analyze. Here, we present a number of statistics for RNA molecular-structure prediction. Besides common pair-wise comparisons, we consider a few reasonable statistics for base-triple predictions, and present an elaborate analysis of these methods. All these statistics incorporate phylogenetic relationships of the sequences in the analysis to varying degrees, and the different nature of these tests gives a wide choice of statistical tools for RNA structure prediction. RESULTS: Starting from statistics that incorporate phylogenetic information only as independent sequence evolution models for each position of a multiple alignment, and extending this idea to a joint evolution model of two positions, we enhance the usual purely statistical methods (e.g. methods based on the Mutual Information statistic) with the use of phylogenetic information available in the sequences. In particular, we present a joint model based on the HKY evolution model, and consequently a X(2) test of independence for two positions. A significant part of this work is devoted to some mathematical analysis of these methods. We tested these statistics on regions of 16S and 23S rRNA, and tRNA.

Base Sequence↗
PubMed2001

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure.

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.

3' Untranslated Regions↗
PubMed2005

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication.

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

3' Untranslated Regions↗
PubMed2003

The common and the distinctive features of the bulged-G motif based on a 1.04 A resolution RNA structure.

Bulged-G motifs are ubiquitous internal RNA loops that provide specific recognition sites for proteins and RNAs. To establish the common and distinctive features of the motif we determined the structures of three variants and compared them with related structures. The variants are 27-nt mimics of the sarcin/ricin loop (SRL) from Escherichia coli 23S ribosomal RNA that is an essential part of the binding site for elongation factors (EFs). The wild-type SRL has now been determined at 1.04 A resolution, supplementing data obtained before at 1.11 A and allowing the first calculation of coordinate error for an RNA motif. The other two structures, having a viable (C2658U*G2663A) or a lethal mutation (C2658G*G2663C), were determined at 1.75 and 2.25 A resolution, respectively. Comparisons reveal that bulged-G motifs have a common hydration and geometry, with flexible junctions at flanking structural elements. Six conserved nucleotides preserve the fold of the motif; the remaining seven to nine vary in sequence and alter contacts in both grooves. Differences between accessible functional groups of the lethal mutation and those of the viable mutation and wild-type SRL may account for the impaired elongation factor binding to ribosomes with the C2658G*G2663C mutation and may underlie the lethal phenotype.

Animals↗
PubMed1998

RNase P RNA structure and cleavage reflect the primary structure of tRNA genes.

The function of RNase P RNA depends on its folding in space. A majority of RNase P RNAs from various bacteria show a similar secondary structure to that of Escherichia coli (M1 RNA). However, there are exceptions as exemplified by the RNase P RNA derived from the low GC-content Gram-positive bacteria Bacillus subtilis and Mycoplasma hyopneumoniae (Hyo P RNA). Previous studies using M1 RNA and Hyo P RNA suggest differences both with respect to the kinetics of cleavage as well as to cleavage site recognition. Here we have studied cleavage by these two structurally different RNase P RNAs as a function of changes in the 5' leader and the 3'-terminal CCA motif in the substrate. Our data suggest that the nucleotide at the -2 position in the 5' leader plays a role both for cleavage site recognition and for the rate of cleavage. However, depending on the identity of the -2 residue differences in the cleavage pattern comparing these two types of RNase P RNAs were observed. The results also suggest that the identity of the -1/+73 base-pair in the substrate influences the cleavage site recognition process. These findings will be related to differences in structure comparing these types of RNase P RNAs and the "RCCA-RNase P RNA" interaction. In addition, our findings will be discussed with respect to the primary structure of the tRNA genes in different bacteria.

Base Sequence↗
PubMed1995

Successful transformation of yeast mitochondria with RPM1: an approach for in vivo studies of mitochondrial RNase P RNA structure, function and biosynthesis.

Mitochondrial RNase P RNA (Rpm1r) is coded by the RPM1 gene of mitochondrial DNA in many yeasts. As an initial step to developing a genetic approach to the structure and biogenesis of yeast mitochondrial RNase P, biolistic transformation has been used to introduce wild type and altered RPM1 genes into strains containing no mitochondrial DNA. The introduced wild type gene does support RNase P activity demonstrating that pre-existing RNase P activity is not necessary for the biosynthesis of the enzyme. Mutations introduced into RPM1 in vitro result in reduced accumulation of mature tRNA and in an alteration of the processing of Rpm1r in vivo.

Base Sequence↗
PubMed1999

A phylogenetic approach to RNA structure prediction.

Methods based on the Mutual Information statistic (MI methods) predict structure by looking for statistical correlations between sequence positions in a set of aligned sequences. Although MI methods are often quite effective, these methods ignore the underlying phylogenetic relationships of the sequences they analyze. Thus, they cannot distinguish between correlations due to structural interactions, and spurious correlations resulting from phylogenetic history. In this paper, we introduce a method analogous to MI that incorporates phylogenetic information. We show that this method accurately recovers the structures of well-known RNA molecules. We also demonstrate, with both real and simulated data, that this phylogenetically-based method outperforms standard MI methods, and improves the ability to distinguish interacting from non-interacting positions in RNA. This method is flexible, and may be applied to the prediction of protein structure given the appropriate evolutionary model. Because this method incorporates phylogenetic data, it also has the potential to be improved with the addition of more accurate phylogenetic information, although we show that even approximate phylogenies are helpful.

Computer Simulation↗
PubMed2002

A Non-sequence-specific double-stranded RNA structural element regulates splicing of two mutually exclusive exons of fibroblast growth factor receptor 2 (FGFR2).

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) mutually exclusive exons IIIb and IIIc represents a tightly regulated and functionally relevant example of post-transcriptional gene regulation. Rat prostate cancer DT3 and AT3 cell lines demonstrate exclusive selection of either exon IIIb or exon IIIc, respectively, and have been used to characterize regulatory FGFR2 RNA cis-elements that are required for splicing regulation. Two sequences termed ISE-2 and ISAR are located in the intron between exons IIIb and IIIc and are required for cell-type specific exon IIIb. Previous studies suggest that the function of these elements involves formation of an RNA stem structure, even though they are separated by more than 700 nucleotides. Using transfected minigenes, we performed a systematic analysis of the sequence and structural components of ISE-2 and ISAR that are required for their ability to regulate FGFR2 splicing. We found that the primary sequence of these elements can be replaced by completely unrelated sequences, provided that they are also predicted to form an RNA stem structure. Thus, a nonsequence-specific double stranded RNA stem constitutes a functional element required for FGFR2 splicing; suggesting that a double-stranded RNA binding protein is a component of the splicing regulatory machinery.

Alternative Splicing↗
PubMed2004

First snapshots of the HIV-1 RNA structure in infected cells and in virions.

With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA(3) (Lys) annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.

5' Untranslated Regions↗
PubMed1999

3'-Terminal RNA structures and poly(U) tracts inhibit initiation by a 3'-->5' exonuclease in vitro.

We have previously shown that the presence of a poly(A) tail blocks the activity of a highly efficient 3'-->5' exonuclease in HeLa extracts. Similar activities have been implicated in RNA turnover in vivo. It is not clear, however, what protects poly(A)-non-mRNAs from the action of this enzyme. A stem-loop structure located at the 3'-end of U11 RNA was required to protect this transcript from the exonuclease in vitro. Similar 3' stem-loop structures, or extensive base pairinginvolving the 3'-end, are present on all mature small stable RNAs. The placement of artificial stem-loop structures at the 3'-end also protected RNA substrates, suggesting that RNA structure alone is sufficient to block the initiation of the exonuclease. The placement of RNA structures at internal positions of substrate trans-cripts did not affect the activity of the exonuclease or lead to the accumulation of degradation intermediates. Pol III precursor transcripts contain short poly(U) tracts rather than structure at their 3'-ends. Terminal poly(U) tracts protected RNA substrates from the 3'-->5' exonuclease in a protein-dependent fashion. Although La protein is found associated with the terminal U tracts of pol III precursor transcripts both in vivo and in vitro, La protein was not required for poly(U) to protect RNA substrates from the 3'-->5' exonuclease. In summary, these data reveal a variety of ways RNAs have evolved to protect themselves from this exonuclease.

3' Untranslated Regions↗
PubMed1989

The completed sequence of lymphocytic choriomeningitis virus reveals a unique RNA structure and a gene for a zinc finger protein.

The arenavirus, lymphocytic choriomeningitis virus (LCMV) has a single-stranded RNA genome composed of a large (L) and a small (S) RNA segment. The completed sequence of LCMV, presented here, reveals a formerly unknown gene (Z) on the L genomic segment. This gene is encoded in the positive or message-sense of the viral genomic RNA, whereas the adjacent gene (L) is in the genome-complementary, or negative sense. The ambisense polarity of the genes on the L RNA reiterates the polarity of genes on the small (S) genomic segment. The Z gene encodes a 10-kDa protein containing a single zinc-finger sequence (Cys2His2). A small RNA representing the message sense of the Z gene is found in infected cells and within virions. In contrast to the known LCMV proteins having structural or enzymatic functions, the predicted Z gene product is most likely to be an RNA-binding protein with a regulatory role. The encapsidation of a message sense Z RNA suggests a role for this gene immediately following virus penetration. The L/Z intergenic region is rich in cytidylic acid (C) and presents an unusual RNA structure. All cDNA clones of the intergenic region differ from each other within a certain poly(C) stretch and lack a 30-base region present in the direct RNA sequence. Finally, the completed sequence establishes that the L RNA 5' end is complementary to its 3' end. The L RNA termini, similar to the S RNA termini, have a small but potentially important asymmetry of sequence. LCMV is the first arenavirus to be completely sequenced.

Amino Acid Sequence↗
PubMed1995

Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication.

The TAR element is a viral regulatory element extending from +1 to +60 in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, which is critical for activation by the transactivator protein Tat. Jurkat cell lines chronically infected with viruses containing HIV-1 TAR element mutations are extremely defective for both gene expression and replication. We previously demonstrated that viruses containing mutations of the TAR RNA stem, bulge, or loop structures have 200- to 5,000-fold-reduced levels of gene expression compared with lymphoid cells harboring wild-type virus. In this study, we characterized several Jurkat cell lines infected with TAR element mutant viruses which spontaneously produced culture supernatants with wild-type-like levels of reverse transcriptase activity. These viral supernatants were used to infect Jurkat cells, and following PCR amplification of the viral long terminal repeats, their DNA sequences were analyzed. This analysis demonstrated that revertant viruses isolated from these cell lines retained the original TAR mutations but also contained additional compensatory mutations within TAR. In gel retardation analysis, recombinant Tat protein bound to higher levels to in vitro-transcribed revertant TAR RNAs than the original TAR RNA mutants. Both the original and revertant TAR elements were inserted into both chloramphenicol acetyltransferase reporter and HIV-1 proviral constructs and assayed following transfection of Jurkat cells. Constructs containing revertant TAR element mutations were capable of strong activation by Tat in contrast to constructs containing the original TAR mutations. Analysis of the secondary structure of TAR RNA sequences suggested that TAR RNA structures which differed from that of wild-type TAR were still capable of strong activation in response to Tat. These results further define critical sequences in TAR RNA that are required for tat activation. In addition, since TAR structures with lower free energy that preserve the loop and bulge structures may be favored over fully formed TAR RNA with higher stable free energy, these results implicate nascent RNA rather than the fully formed TAR RNA structure as the target for tat activation.

Base Sequence↗