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In many cases RNA can assume co-existing or meta-stable structures preventing structure determination by chemical mapping. A novel method is described, by which RNA is modified with dimethyl sulphate without shifting the distribution of different structures. The different structures are then separated in native gel electrophoresis, and structure determination by primer extension can be carried out separately for each structure.
Human RNA polymerase II recognizes a strong transcriptional pause signal in the initially transcribed region of HIV-1. We report the use of a limited-step transcription assay to dissect the mechanism underlying recognition of and escape from this HIV-1 pause. Our results suggest that the primary determinant of transcriptional pausing is a relatively weak RNA:DNA hybrid that triggers backtracking of RNA polymerase II along the RNA and DNA chains and displaces the RNA 3' OH from the active site. In contrast, two alternative RNA secondary structures, TAR and anti-TAR, are not required for pausing and affect it only indirectly, rather than through direct interaction with RNA polymerase II. TAR accelerates escape from the pause, but anti-TAR inhibits formation of TAR prior to pause escape. The behavior of RNA polymerase II at a mutant pause signal supports a two-step, non-equilibrium mechanism in which the rate-determining step is a conformational change in the enzyme, rather than the changes in nucleic-acid base-pairing that accompany backtracking.
RNA secondary and tertiary structure is involved in post-transcriptional regulation of gene expression either by exposing specific sequences or through the formation of specific structural motifs. An overview of RNA secondary and tertiary structures known from biophysical studies is followed by a review of examples of the elements of RNA processing, mRNA stability and translation of the messenger. These structural elements comprise sense-antisense double-stranded RNA, hairpin and stem-loop structures, and more complex structures such as bifurcations, pseudoknots and triple-helical elements. Metastable structures formed during RNA folding pathway are also discussed. The examples presented are mostly chosen from plant systems, plant viruses, and viroids. Examples from bacteria or fungi are discussed only when unique regulatory properties of RNA structures have been elucidated in these systems.
BACKGROUND: It has become increasingly apparent that a comprehensive database of RNA motifs is essential in order to achieve new goals in genomic and proteomic research. Secondary RNA structures have frequently been represented by various modeling methods as graph-theoretic trees. Using graph theory as a modeling tool allows the vast resources of graphical invariants to be utilized to numerically identify secondary RNA motifs. The domination number of a graph is a graphical invariant that is sensitive to even a slight change in the structure of a tree. The invariants selected in this study are variations of the domination number of a graph. These graphical invariants are partitioned into two classes, and we define two parameters based on each of these classes. These parameters are calculated for all small order trees and a statistical analysis of the resulting data is conducted to determine if the values of these parameters can be utilized to identify which trees of orders seven and eight are RNA-like in structure. RESULTS: The statistical analysis shows that the domination based parameters correctly distinguish between the trees that represent native structures and those that are not likely candidates to represent RNA. Some of the trees previously identified as candidate structures are found to be "very" RNA like, while others are not, thereby refining the space of structures likely to be found as representing secondary RNA structure. CONCLUSION: Search algorithms are available that mine nucleotide sequence databases. However, the number of motifs identified can be quite large, making a further search for similar motif computationally difficult. Much of the work in the bioinformatics arena is toward the development of better algorithms to address the computational problem. This work, on the other hand, uses mathematical descriptors to more clearly characterize the RNA motifs and thereby reduce the corresponding search space. These preliminary findings demonstrate that graph-theoretic quantifiers utilized in fields such as computer network design hold significant promise as an added tool for genomics and proteomics.
A combination of algorithms to search RNA sequence for the potential for secondary structure formation, and search large numbers of sequences for structural similarity, were used to search the 5'UTRs of annotated genes in the Escherichia coli genome for regulatory RNA structures. Using this approach, similar RNA structures that regulate genes in the thiamin metabolic pathway were identified. In addition, several putative regulatory structures were discovered upstream of genes involved in other metabolic pathways including glycerol metabolism and ethanol fermentation. The results demonstrate that this computational approach is a powerful tool for discovery of important RNA structures within prokaryotic organisms.
The RNA challenge phage system enables genetic selection of proteins with RNA-binding activity in bacteria. These phages are modified versions of the temperate DNA bacteriophage P22 in which post-transcriptional regulatory events control the developmental fate of the phage. The system was originally developed to identify novel RNA ligands that display reduced affinity for the R17/MS2 coat protein, as well as to select for suppressor coat proteins that recognize mutant RNA ligands. During the course of evaluating whether the HIV-1 Rev protein could direct lysogen development for bacteriophage derivatives that encode Rev response element (RRE) RNA sequences, two examples of RRE RNA ligands that interfere with challenge phage development were identified. In the phage examples described, RRE RNA secondary structure prevents Ant protein biosynthesis and lytic development. Phage lysogen formation occurs efficiently in recipient cells, independent of the expression status of the Rev protein or trans-acting competitor RRE RNA ligands. These studies provide the first example whereby RNA challenge phages may be applied to study RNA folding events and RNA structural interactions in an in vivo context.
Proteins interacting with RNA structures at the 3' non-translated region (3'NTR) of picornaviruses are probably important during viral RNA replication. We have shown previously that a dominant cellular cytoplasmic protein of 38 kDa (p38) interacts with the 3'NTR and upstream regions of the hepatitis A virus (HAV) RNA (Kusov et al., J Virol 70, 1890-1897, 1996). Immunological and biochemical analyses of p38 have indicated that it is identical to GAPDH, which has previously been described as modulating translational regulation of the HAV RNA by interacting with the 5'NTR (Schultz et al., J Biol Chem 271, 14134-14142, 1996). Three separate binding regions for GAPDH in the 3'NTR and in the upstream 3D polymerase-coding region were identified. Structural analysis of these RNA regions by computer modelling and direct enzymatic cleavage suggested the presence of several AU-rich stem-loop structures having the potential for tertiary interactions. Binding of GAPDH to these structures was confirmed by RNA footprint analysis and resulted in the loss of double-stranded RNA regions. A different panel of RNA binding proteins (p28, p41 and p65) was detected in the ribosomal fractions of several cell lines (BSC-1, FRhK-4 and HeLa), whereas RNA binding of the GAPDH that was also present in these fractions was only marginal or absent.
BACKGROUND: gene identification in genomic DNA sequences by computational methods has become an important task in bioinformatics and computational gene prediction tools are now essential components of every genome sequencing project. Prediction of splice sites is a key step of all gene structural prediction algorithms. RESULTS: we sought the role of mRNA secondary structures and their information contents for five vertebrate and plant splice site datasets. We selected 900-nucleotide sequences centered at each (real or decoy) donor and acceptor sites, and predicted their corresponding RNA structures by Vienna software. Then, based on whether the nucleotide is in a stem or not, the conventional four-letter nucleotide alphabet was translated into an eight-letter alphabet. Zero-, first- and second-order Markov models were selected as the signal detection methods. It is shown that applying the eight-letter alphabet compared to the four-letter alphabet considerably increases the accuracy of both donor and acceptor site predictions in case of higher order Markov models. CONCLUSION: Our results imply that RNA structure contains important data and future gene prediction programs can take advantage of such information.
Terminal transferase-dependent PCR (TDPCR) can be used after reverse transcription to analyze RNA. This method (RT-TDPCR) is able to provide in vivo information at nucleotide-level resolution, and has been used for study of ribozymes, RNA size, RNA structure, and RNA-protein interactions. A detailed protocol of RT-TDPCR is presented here with examples of its use in detecting ribozyme cleavage intermediates in yeast and a RNA transcription start site in mammalian cells.
Ab initio RNA secondary structure predictions have long dismissed helices interior to loops, so-called pseudoknots, despite their structural importance. Here we report that many pseudoknots can be predicted through long-time-scale RNA-folding simulations, which follow the stochastic closing and opening of individual RNA helices. The numerical efficacy of these stochastic simulations relies on an O(n2) clustering algorithm that computes time averages over a continuously updated set of n reference structures. Applying this exact stochastic clustering approach, we typically obtain a 5- to 100-fold simulation speed-up for RNA sequences up to 400 bases, while the effective acceleration can be as high as 105-fold for short, multistable molecules (<or=150 bases). We performed extensive folding statistics on random and natural RNA sequences and found that pseudoknots are distributed unevenly among RNA structures and account for up to 30% of base pairs in G+C-rich RNA sequences (online RNA-folding kinetics server including pseudoknots: http://kinefold.u-strasbg.fr).
Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5' splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5' ss of exon 7. We call this structure terminal stem-loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5' ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.
Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA poly-adenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3'-ends of most of the mRNA molecules in bacteria are characterized by a stem-loop structure. We show here that in vitro PAP I activity is inhibited by a stem-loop structure. A tail of two to six nucleo-tides located 3' to the stem-loop structure is sufficient to overcome this inhibition. These results suggest that the stem-loop structure located in most of the mRNA 3'-ends may function as an inhibitor of poly-adenylation and degradation of the corresponding RNA molecule. However, RNA 3'-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.
Cobalamin in the form of adenosylcobalamin (Ado-CBL) is known to repress expression of genes for vitamin B(12) biosynthesis and be transported by a posttranscriptional regulatory mechanism, which involves direct binding of Ado-CBL to 5'untranslated gene regions (5'UTR). Using comparative analysis of genes and regulatory regions, we identified a highly conserved RNA structure, the B12-element, which is widely distributed in 5'UTRs of vitamin B(12)-related genes in eubacteria. Multiple alignment of approximately 200 B12-elements from 66 bacterial genomes reveals their common secondary structure and several extended regions of sequence conservation, including the previously known B12-box motif. In analogy to the model of regulation of the riboflavin and thiamin biosynthesis, we suggest Ado-CBL-mediated regulation based on formation of alternative RNA structures including the B12-element. In Gram-negative proteobacteria, as well as in cyanobacteria, actinobacteria, and the CFB group, the cobalamin biosynthesis and vitamin B(12) transport genes are predicted to be regulated by inhibition of translation initiation, whereas in the Bacillus/Clostridium group of Gram-positive bacteria, these genes seem to be regulated by transcriptional antitermination. Phylogenetic analysis of the B12-elements reveals a large number of likely duplications of B12-elements in several bacterial genomes. These lineage-specific duplications of RNA regulatory elements seem to be a major evolutionary mechanism for expansion of the vitamin B(12) regulon.
Antisense RNA-mediated regulation in bacterial systems is related to the kinetics of RNA-RNA annealing in vitro. Here, we investigated the secondary structure of alphaY69, an effective HIV-directed antisense RNA in human cells. Purified RNA preparations contain a single conformer. The global structure was identified by a cleavage experiment under native conditions using a short complementary oligonucleotide and RNase H. Structural analyses indicate a three-domain structure of alphaY69 consisting of two stem-loop elements connected by a seven-nucleotide single-stranded hinge region. Kinetic data suggest that the formation of base pairs between a CGC triplet of alphaY69 and its target RNA is essential for fast annealing. The complementary sequence stretch of the target folds into a high-energy secondary structure. The relationship between modifications in structural elements of alphaY69 and the annealing kinetics suggested that rate-limiting steps of the annealing involve a single site of alphaY69 and do not involve its 5' or 3'-end. Further, the data indicate that both initial base-specific interactions and duplex formation are dependent on the CGC triplet of the central region of alphaY69. This mechanism represents a specific and efficient way of RNA-RNA annealing that is initiated by the interaction of unstructured RNA regions.
The telomerase ribonucleoprotein has a phylogenetically divergent RNA subunit, which contains a short template for telomeric DNA synthesis. To understand how telomerase RNA participates in mechanistic aspects of telomere synthesis, we studied a conserved secondary structure adjacent to the template. Disruption of this structure caused DNA synthesis to proceed beyond the normal template boundary, resulting in altered telomere sequences, telomere shortening, and cellular growth defects. Compensatory mutations restored normal telomerase function. Thus, the RNA structure, rather than its sequence, specifies the template boundary. This study reveals a specific function for an RNA structure in the enzymatic action of telomerase.
Reverse transcription of the RNA genome of retroviruses has to proceed through some highly structured regions of the template. The RNA genome of the human immunodeficiency virus type 1 (HIV-1) contains two hairpin structures within the repeat (R) region at the 5' end of the viral RNA (Fig. 1Fig. 1Template RNA structure of the HIV-1 R region and the position of reverse transcription pause sites. The HIV-1 R region (nucleotides +1/97) encodes two stable RNA structures, the TAR and polyA hairpins [5]. The latter hairpin contains the AAUAAA hexamer motif (marked by a box) that is involved in polyadenylation. The lower panel shows the predicted structures of the wild-type and two mutant forms of the polyA hairpin that were used in this study. Nucleotide substitutions are boxed, deletions are indicated by black triangle. The thermodynamic stability (free energy or DeltaG, in kcal/mol) was calculated according to the Zucker algorithm [71]. The TAR hairpin has a DeltaG of -24.8 kcal/mol. Minus-strand DNA synthesis on these templates was initiated by a DNA primer annealed to the downstream PBS. The position of reverse transcription pause sites observed in this study are summarized. All numbers refer to nucleotide positions on the wild-type HIV-1 transcript. Filled arrows represent stops observed on the wild-type template, and open arrows mark the pause sites that are specific for the structured A-mutant template. The sizes of the arrows correspond to the relative frequency of pausing. Little pausing was observed on the B-mutant template with the destabilized polyA hairpin.). These structures, the TAR and polyA hairpins, fulfil important functions in the viral life cycle. We analyzed the in vitro elongation properties of the HIV-1 reverse transcriptase (RT) enzyme on the wild-type RNA template and mutants thereof with either a stabilized or a destabilized polyA hairpin. Stable RNA structure was found to interfere with efficient elongation of the RT enzyme, as judged by the appearance of pause cDNA products. A direct relation was measured between the stability of template RNA structure and the extent of RT pausing. However, the position of structure-induced pause sites is rather diverse, with significant stops at a position approximately 6 nt ahead of the basepaired stem of the TAR and polyA hairpins. This suggests that the RT enzyme is stalled when its most forward domain contacts the RNA duplex. Addition of the viral nucleocapsid protein (NC) to the in vitro assay was found to overcome such structure-induced RT stops. These results indicate that the RT polymerase has problems penetrating regions of the template with stable RNA structure. This effect was more pronounced at high Mg2+ concentrations, which is known to stabilize RNA secondary structure. Such a structure-induced defect was not apparent in reverse transcription assays performed in virus-infected cells, which is either caused by the NC protein or other components of the virion particle. Thus, retroviruses can use relatively stable RNA structures to control different steps in the viral life cycle without interfering with the process of reverse transcription.
BACKGROUND: An increasing number of researchers have released novel RNA structure analysis and prediction algorithms for comparative approaches to structure prediction. Yet, independent benchmarking of these algorithms is rarely performed as is now common practice for protein-folding, gene-finding and multiple-sequence-alignment algorithms. RESULTS: Here we evaluate a number of RNA folding algorithms using reliable RNA data-sets and compare their relative performance. CONCLUSIONS: We conclude that comparative data can enhance structure prediction but structure-prediction-algorithms vary widely in terms of both sensitivity and selectivity across different lengths and homologies. Furthermore, we outline some directions for future research.
By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.