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At least 991 records · Page 55Linked to original sources
PubMed2005

Pseudouridine modification in Caenorhabditis elegans spliceosomal snRNAs: unique modifications are found in regions involved in snRNA-snRNA interactions.

BACKGROUND: Pseudouridine (Psi) is an abundant modified nucleoside in RNA and a number of studies have shown that the presence of Psi affects RNA structure and function. The positions of Psi in spliceosomal small nuclear RNAs (snRNAs) have been determined for a number of species but not for the snRNAs from Caenorhabditis elegans (C. elegans), a popular experimental model system of development. RESULTS: As a prelude to determining the function of or requirement for this modification in snRNAs, we have mapped the positions of Psi in U1, U2, U4, U5, and U6 snRNAs from worms using a specific primer extension method. As with other species, C. elegans U2 snRNA has the greatest number of Psi residues, with nine, located in the 5' half of the U2 snRNA. U5 snRNA has three Psis, in or near the loop of the large stem-loop that dominates the structure of this RNA. U6 and U1 snRNAs each have one Psi, and two Psi residues were found in U4 snRNA. CONCLUSION: The total number of Psis found in the snRNAs of C. elegans is significantly higher than the minimal amount found in yeasts but it is lower than that seen in sequenced vertebrate snRNAs. When the actual sites of modification on C. elegans snRNAs are compared with other sequenced snRNAs most of the positions correspond to modifications found in other species. However, two of the positions modified on C. elegans snRNAs are unique, one at position 28 on U2 snRNA and one at position 62 on U4 snRNA. Both of these modifications are in regions of these snRNAs that interact with U6 snRNA either in the spliceosome or in the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and the presence of Psi may be involved in strengthening the intermolecular association of the snRNAs.

Animals↗
PubMed1995

GNRA tetraloops make a U-turn.

The U-turn (uridine turn) is an RNA structural motif that contains a change in backbone direction stabilized by specific interactions across the bend. It was first identified in the anticodon loop and the T-loop of yeast tRNA(Phe) (Quigley & Rich, 1976, Science 194:796-806) and has recently also been found in the crystal structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB, 1994a, Nature 372:68-74). These U-turn motifs follow a UNR consensus sequence (where N is any nucleotide and R is G or A). Here we report that the frequently occurring GNRA tetraloops also contain a U-turn motif, and we discuss the role of U-turns as abundant tertiary structural motifs in RNA.

Nucleic Acid Conformation↗
PubMed1989

RNA secondary structure is an integral part of the in vitro mechanism of attenuation in simian virus 40.

At late times after infection with SV40, a prematurely terminated transcript that initiates at the major late promoter (MLP) and has a 3'-end about 95 nucleotides downstream has been identified and termed an attenuated RNA (Hay, N., Skolnik-David, H., and Aloni, Y. (1982) Cell 29, 183-193). The DNA template of the attenuated RNA has two regions of dyad symmetry, and the attenuated RNA can therefore fold into two hairpin elements. The hairpin element at the 3'-end of the attenuated RNA is followed by a stretch of Us and resembles a rho-independent terminator in prokaryotes. We have suggested that folding of the RNA into two hairpin elements will lead to a block of transcription elongation. Using site-directed mutagenesis, we created two templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared between the templates in a cell-free extract. We have shown that RNA polymerase II recognizes the SV40 sequence that leads to a block of transcription elongation, even when it is under the control of the MLP of adenovirus 2. The extent of the block of transcription elongation is directly dependent on the stability of the hairpin structure of the RNA as assessed by a comparison of transcription of the wild-type and mutated templates. The addition of Sarkosyl and transcription at an elevated temperature during the elongation reaction enhanced the production of the attenuated RNA from all templates.

Base Sequence↗
PubMed1984

Alterations of [3H]actinomycin D binding to axotomized dorsal root ganglion cell nuclei: an autoradiographic method to detect changes in chromatin structure and RNA synthesis.

An autoradiographic method was developed to quantify on a comparative basis the binding of [3H]actinomycin D (Act D) to the cell nuclei of frozen, unfixed sections of spinal sensory ganglia in rats. After a crush lesion of the sciatic nerve, alterations of [3H]Act D binding were found in L5 and L6 dorsal root ganglia which corresponded to changes in RNA synthesis observed in other studies. An increase in Act D binding was seen at 1 to 3 days postoperation, followed by a decrease at 5 to 7 days. By 9 to 11 days a second increase in binding occurred, followed by a decrease at 14 days. Contralateral ganglia exhibited an increase in Act D binding only at 5 days compared with unoperated controls. The timing of the response in axotomized ganglia differed with the distance of the lesion from the cell body. The observed patterns of Act D binding confirm that changes of chromatin structure are closely associated with the alterations of RNA and protein synthesis occurring after axon injury. The method may be useful as an indicator for alterations in RNA synthesis related to changes in chromatin structure in complex tissues.

Animals↗
PubMed1999

Nuclear snRNA and nuclear function (discovery of 5' cap structures in RNA).

In view of the fact that eukaryotic gene expression starts in the nucleus, it is important to have a thorough understanding of nuclear macromolecular structure and function. Newly discovered snRNAs are eukaryotic cell specific and have unique subnuclear compartmental localizations. There are over 200 nucleolar-specific RNAs that include some abundant U3, U8, and U13 RNAs. Extranucleolar-nuclear-specific RNAs (snRNA) are 4.5S RNA I, II, III, 5S RNA III, U1, U2, U4, U5, and U6, in addition to over 500 different RNA species reported up to now. In particular, some snoRNAs and snRNAs have trimethylguanosine cap structures that are not present in bacteria. They have crucial roles in gene expression, such as transcription (U3 snoRNA), processing (U3, U8, U13, U14, U22, and 7-2/MRP), methylation (U14-16, U18, U20-21, and U24-63), pseudouridylation (E2, E3, U19, U23, and U64-72), and hnRNA splicing (U1, U2, U4, U5, and U6 snRNA).

Base Sequence↗
PubMed2001

TFIIH inhibits CDK9 phosphorylation during human immunodeficiency virus type 1 transcription.

Tat stimulates human immunodeficiency virus, type 1 (HIV-1), transcription elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to the TAR RNA structure. It has been demonstrated further that CDK9 phosphorylation is required for high affinity binding of Tat/P-TEFb to the TAR RNA structure and that the state of P-TEFb phosphorylation may regulate Tat transactivation. We now demonstrate that CDK9 phosphorylation is uniquely regulated in the HIV-1 preinitiation and elongation complexes. The presence of TFIIH in the HIV-1 preinitiation complex inhibits CDK9 phosphorylation. As TFIIH is released from the elongation complex between +14 and +36, CDK9 phosphorylation is observed. In contrast to the activity in the "soluble" complex, phosphorylation of CDK9 is increased by the presence of Tat in the transcription complexes. Consistent with these observations, we have demonstrated that purified TFIIH directly inhibits CDK9 autophosphorylation. By using recombinant TFIIH subcomplexes, our results suggest that the XPB subunit of TFIIH is responsible for this inhibition of CDK9 phosphorylation. Interestingly, our results further suggest that the phosphorylated form of CDK9 is the active kinase for RNA polymerase II carboxyl-terminal domain phosphorylation.

Blotting, Western↗
PubMed1998

Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies.

BACKGROUND: The translation or stability of the mRNAs from ferritin, maconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of high-resolution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms. RESULTS: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern. CONCLUSIONS: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes. The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.

Animals↗
PubMed1984

DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome.

A complementary DNA clone of 7 SK RNA from HeLa cells was used to study the genomic organization of 7 SK sequences in the human genome. Genomic hybridizations and genomic clones show that 7 SK is homologous to a family of disperse repeated sequences most of which lack the 3' end of the 7 SK RNA sequence. Only few of the genomic K sequences are homologous to both 3' and 5' 7 SK probes and presumably include the gene(s) for 7 SK RNA. The sequence of four genomic 7 SK clones confirms that they are in most cases pseudogenes. Although Alu sequences are frequently found near the 3' and 5' end of K DNA, the sequences immediately flanking the pseudogenes are different in all clones studied. However, direct repeats were found flanking directly the K DNA or the K-Alu unit, suggesting that the K sequences alone or in conjunction with Alu DNA might constitute a mobile element.

Base Sequence↗
PubMed2002

Genetic perturbations of RNA reveal structure-based recognition in protein-RNA interaction.

Protein-RNA recognition is an essential foundation of cellular processes, yet much remains unknown about these important interactions. The recognition between aminoacyl-tRNA synthetases and their cognate tRNA substrates is highly specific and essential for cell viability, due to the necessity for accurate translation of the genetic code into protein sequences. We selected an active tRNA that is highly mutated in the recognition nucleotides of the acceptor stem region in the alanine system. The functional properties of this mutant and its secondary derivatives demonstrate that recognition cannot be reduced to isolated structural elements, but rather the amino acid acceptor stem is being recognized as a unit.

Amino Acyl-tRNA Synthetases↗
PubMed2004

A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication.

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.

Base Sequence↗
PubMed1999

Mapping of protein domains of hepatitis A virus 3AB essential for interaction with 3CD and viral RNA.

The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.

3C Viral Proteases↗
PubMed1999

Changes in Rous sarcoma virus RNA secondary structure near the primer binding site upon tRNATrp primer annealing.

Predicted secondary-structure elements encompassing the primer binding site in the 5' untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464-2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648-7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TPsiC interaction region. Limited digestion of the 5' untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TPsiC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

Animals↗
PubMed2004

Co-transcriptional folding is encoded within RNA genes.

BACKGROUND: Most of the existing RNA structure prediction programs fold a completely synthesized RNA molecule. However, within the cell, RNA molecules emerge sequentially during the directed process of transcription. Dedicated experiments with individual RNA molecules have shown that RNA folds while it is being transcribed and that its correct folding can also depend on the proper speed of transcription. METHODS: The main aim of this work is to study if and how co-transcriptional folding is encoded within the primary and secondary structure of RNA genes. In order to achieve this, we study the known primary and secondary structures of a comprehensive data set of 361 RNA genes as well as a set of 48 RNA sequences that are known to differ from the originally transcribed sequence units. We detect co-transcriptional folding by defining two measures of directedness which quantify the extend of asymmetry between alternative helices that lie 5' and those that lie 3' of the known helices with which they compete. RESULTS: We show with statistical significance that co-transcriptional folding strongly influences RNA sequences in two ways: (1) alternative helices that would compete with the formation of the functional structure during co-transcriptional folding are suppressed and (2) the formation of transient structures which may serve as guidelines for the co-transcriptional folding pathway is encouraged. CONCLUSIONS: These findings have a number of implications for RNA secondary structure prediction methods and the detection of RNA genes.

Algorithms↗
PubMed2003

Primary sequence and secondary structure motifs in spleen necrosis virus RU5 confer translational utilization of unspliced human immunodeficiency virus type 1 reporter RNA.

The 5' long terminal repeat (LTR) of spleen necrosis virus (SNV) contains a unique posttranscriptional control element that facilitates Rev/Rev-responsive element-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA. HIV-1 Gag expression is eliminated when SNV LTR is repositioned to the 3' untranslated region or when the RU5 region is positioned in the antisense orientation. RU5 corresponds to the 5' RNA terminus, and results presented here indicate that Gag production is sustained upon introduction of transcribed spacers that reposition SNV RU5 35 to 200 nucleotides downstream. Concordant results of deletion and point mutagenesis identified two functionally redundant and synergistic motifs (designated A and C) that are necessary and sufficient for SNV RU5 activity. Enzymatic analysis of SNV RU5 RNA structure determined that A and C correspond to stem-loop structures. Quantitative RNA and protein analysis of A and C mutants revealed that the structural integrity of A and C is necessary for protein production, and loss of function correlates with little change in steady-state level, splicing efficiency, or cytoplasmic accumulation of HIV-1 gag reporter RNA. Instead, the structural mutations eliminate cytoplasmic utilization as an mRNA template for Gag protein production. Point mutations of unpaired loop-and-bulge nucleotides that maintain the structure of A eliminate activity. The results show that the unpaired UUGU loop and U-rich bulges function together and are candidate SNV RU5 binding sites for the host cell protein(s) that directs cytoplasmic utilization of unspliced HIV-1 reporter RNA.

5' Untranslated Regions↗
PubMed2005

Ribose 2'-F labeling: a simple tool for the characterization of RNA secondary structure equilibria by 19F NMR spectroscopy.

We present a facile 19F NMR spectroscopic approach for the characterization of bistable RNA. The concept is based on the site-specific labeling of RNA with single 2'-deoxy-2'-fluoro (2'-F) nucleosides. Depending on the position of the 2'-F label (either within a double helix or a single-stranded region), significantly different 19F chemical shifts were observed and assigned to the particular alternative folds. This has been exemplified for a series of bistable RNAs. The approach is promising for the detailed investigation of complex RNA folding pathways.

Fluorine↗
PubMed2002

Direct and indirect contributions of RNA secondary structure elements to the initiation of HIV-1 reverse transcription.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition between the viral RNA (vRNA), tRNA(3)(Lys), which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between the HIV-1 RNA and tRNA(3)(Lys). Here, we compared the relative importance of the secondary structure elements of this complex in the initiation process. To this aim, we used the previously published three-dimensional model of the initiation complex to rationally introduce a series of deletions and substitutions in the vRNA. When necessary, we used chemical probing to check the structure of the tRNA(3)(Lys)-mutant vRNA complexes. For each of them, we measured the binding affinity of RT and the kinetics of initial extension of tRNA(3)(Lys) and of synthesis of the (-) strand strong stop DNA. Our results were overall in keeping with the three-dimensional model of the initiation complex. Surprisingly, we found that disruption of the intermolecular template-primer interactions, which are not directly recognized by RT, more severely affected reverse transcription than deletions or disruption of one of the intramolecular helices to which RT directly binds. Perturbations of the highly constrained junction between the intermolecular helix formed by the primer binding site and the 3' end of tRNA(3)(Lys) and the helix immediately upstream also had dramatic effects on the initiation of reverse transcription. Taken together, our results demonstrate the overwhelming importance of the overall three-dimensional structure of the initiation complex and identify structural elements that constitute promising targets for anti-initiation-specific drugs.

Base Sequence↗