The building blocks and motifs of RNA architecture

RNA motifs can be defined broadly as recurrent structural elements containing multiple intramolecular RNA-RNA interactions, as observed in atomic-resolution RNA structures. They constitute the modular building blocks of RNA architecture, which is organized hierarchically. Recent work has focused on analyzing RNA backbone conformations to identify, define and search for new instances of recurrent motifs in X-ray structures. One current view asserts that recurrent RNA strand segments with characteristic backbone configurations qualify as independent motifs. Other considerations indicate that, to characterize modular motifs, one must take into account the larger structural context of such strand segments. This follows the biologically relevant motivation, which is to identify RNA structural characteristics that are subject to sequence constraints and that thus relate RNA architectures to sequences.     

Secondary structure constraints on the evolution of Drosophila 28 S ribosomal RNA expansion segments

Eukaryotic ribosomal RNA genes contain rapidly evolving regions of unknown function termed expansion segments. We present the comparative analysis of the primary and secondary structure of two expansion segments from the large subunit rRNA gene of ten species of Drosophila and the tsetse fly species Glossina morsitans morsitans. At the primary sequence level, most of the differences observed in the sequences obtained are single base substitutions. This is in marked contrast with observations in vertebrate species in which the insertion or deletion of repetitive motifs, probably generated by a DNA-slippage mechanism, is a major factor in the evolution of these regions. The secondary structure of the two regions, supported by multiple compensatory base changes, is highly conserved between the species examined and supports the existence of a general folding pattern for all eukaryotes. Intriguingly, the evolutionary rate of expansion segments is very slow relative to other genic and non-genic regions of the Drosophila genome. These results suggest that the evolution of expansion segments in the rDNA multigene family is a balance between the homogenization of new mutations by unequal crossing over and a combination of selection against some such mutations per se and selection for subsequent compensatory mutations, in order to maintain a particular RNA secondary structure.     

Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs

The set of "expansion segments" of any eukaryotic 26S/28S ribosomal RNA (rRNA) gene is responsible for the bulk of the difference in length between the prokaryotic 23S rRNA gene and the eukaryotic 26S/28S rRNA gene. The expansion segments are also responsible for interspecific fluctuations in length during eukaryotic evolution. They show a consistent bias in base composition in any species; for example, they are AT rich in Drosophila melanogaster and GC rich in vertebrate species. Dot-matrix comparisons of sets of expansion segments reveal high similarities between members of a set within any 28S rRNA gene of a species, in contrast to the little or spurious similarity that exists between sets of expansion segments from distantly related species. Similarities among members of a set of expansion segments within any 28S rRNA gene cannot be accounted for by their base-compositional bias alone. In contrast, no significant similarity exists within a set of "core" segments (regions between expansion segments) of any 28S rRNA gene, although core segments are conserved between species. The set of expansion segments of a 26S/28S gene is coevolving as a unit in each species, at the same time as the family of 28S rRNA genes, as a whole, is undergoing continual homogenization, making all sets of expansion segments from all ribosomal DNA (rDNA) arrays in a species similar in sequence. Analysis of DNA simplicity of 26S/28S rRNA genes shows a direct correlation between significantly high relative simplicity factors (RSFs) and sequence similarity among a set of expansion segments. A similar correlation exists between RSF values, overall rDNA lengths, and the lengths of individual expansion segments. Such correlations suggest that most length fluctuations reflect the gain and loss of simple sequence motifs by slippage-like mechanisms. We discuss the molecular coevolution of expansion segments, which takes place against a background of slippage-like and unequal crossing-over mechanisms of turnover that are responsible for the accumulation of interspecific differences in rDNA sequences.      

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae)

The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5' end (SACUUUUGY) and at the 3' end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.     

Bioinformatic prediction of polymerase elements in the rotavirus VP1 protein

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.     

An algorithm for searching RNA motifs in genomic sequences

RNA molecules, which are found in all living cells, fold into characteristic structures that account for their diverse functional activities. Many of these RNA structures consist of a collection of fundamental RNA motifs. The various combinations of RNA basic components form different RNA classes and define their unique structural and functional properties. The availability of many genome sequences makes it possible to search computationally for functional RNAs. Biological experiments indicate that functional RNAs have characteristic RNA structural motifs represented by specific combinations of base pairings and conserved nucleotides in the loop regions. The searching for those well-ordered RNA structures and their homologues in genomic sequences is very helpful for the understanding of RNA-based gene regulation. In this paper, we consider the following problem: given an RNA sequence with a known secondary structure, efficiently determine candidate segments in genomic sequences that can potentially form RNA secondary structures similar to the given RNA secondary structure. Our new bottom-up approach searches all potential stem-loops similar to ones of the given RNA secondary structure first, and then based on located stem-loops, detects potential homologous structural RNAs in genomic sequences.     

FR3D: finding local and composite recurrent structural motifs in RNA 3D structures

New methods are described for finding recurrent three-dimensional (3D) motifs in RNA atomic-resolution structures. Recurrent RNA 3D motifs are sets of RNA nucleotides with similar spatial arrangements. They can be local or composite. Local motifs comprise nucleotides that occur in the same hairpin or internal loop. Composite motifs comprise nucleotides belonging to three or more different RNA strand segments or molecules. We use a base-centered approach to construct efficient, yet exhaustive search procedures using geometric, symbolic, or mixed representations of RNA structure that we implement in a suite of MATLAB programs, “Find RNA 3D” (FR3D). The first modules of FR3D preprocess structure files to classify base-pair and -stacking interactions. Each base is represented geometrically by the position of its glycosidic nitrogen in 3D space and by the rotation matrix that describes its orientation with respect to a common frame. Base-pairing and base-stacking interactions are calculated from the base geometries and are represented symbolically according to the Leontis/Westhof basepairing classification, extended to include base-stacking. These data are stored and used to organize motif searches. For geometric searches, the user supplies the 3D structure of a query motif which FR3D uses to find and score geometrically similar candidate motifs, without regard to the sequential position of their nucleotides in the RNA chain or the identity of their bases. To score and rank candidate motifs, FR3D calculates a geometric discrepancy by rigidly rotating candidates to align optimally with the query motif and then comparing the relative orientations of the corresponding bases in the query and candidate motifs. Given the growing size of the RNA structure database, it is impossible to explicitly compute the discrepancy for all conceivable candidate motifs, even for motifs with less than ten nucleotides. The screening algorithm that we describe finds all candidate motifs whose geometric discrepancy with respect to the query motif falls below a user-specified cutoff discrepancy. This technique can be applied to RMSD searches. Candidate motifs identified geometrically may be further screened symbolically to identify those that contain particular basepair types or base-stacking arrangements or that conform to sequence continuity or nucleotide identity constraints. Purely symbolic searches for motifs containing user-defined sequence, continuity and interaction constraints have also been implemented. We demonstrate that FR3D finds all occurrences, both local and composite and with nucleotide substitutions, of sarcin/ricin and kink-turn motifs in the 23S and 5S ribosomal RNA 3D structures of the H. marismortui 50S ribosomal subunit and assigns the lowest discrepancy scores to bona fide examples of these motifs. The search algorithms have been optimized for speed to allow users to search the non-redundant RNA 3D structure database on a personal computer in a matter of minutes.     

The genome of Uukuniemi virus consists of three unique RNA segments

The three RNA species isolated from virions of Uukuniemi virus, a proposed member of the newly defined Bunyaviridae family, have been characterized by analysis of ³²P-labeled ribonuclease T1 oligonucleotides separated on two-dimensional polyacrylamide gels. Each RNA species contains unique oligonucleotides not present in the two others, indicating that the genome of this virus is segmented. Each segment appears to contain a unique primary sequence with little or no overlapping among the segments. The complexities of the RNA segments as calculated from the radioactivity in unique oligonucleotides of defined lengths are about 8000 (L RNA), 3500 (M) and 1900 (S) nucleotides. Since these values are similar to the molecular weights determined by other methods, each size class of RNA corresponds to a single molecular species. The presence of a 5′ terminal pppAp … structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed. The absence of either a 5′ terminal “cap” or 3′ terminal poly(A) supports the concept that Uukuniemi virus is a negative strand virus.     

Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein.

It has been widely documented that the nucleocapsid protein p12 (NC) of Rous sarcoma virus (RSV) has a role in the encapsidation and maturation of the virus genomic RNA during particle formation, and particularly important appear to be the Cys-His motifs of this protein. Since some retroviruses only have one such motif, we have investigated the significance of the two distinct Cys-His motifs of RSV NC. The analysis of the phenotype of virus NC mutants with precise rearrangements or duplications of the motifs highlights the following features. (i) The two motifs are not functionally equivalent. (ii) The order and number of Cys-His motifs are less important for RSV NC than the presence of two distinct motifs for both the encapsidation of virus genomic RNA and maintenance of the integrity of the RNA after particle formation. (iii) The proximal motif has a distinct function in the virus replication cycle other than RNA encapsidation and dimerization. (iv) The presence of three Cys-His motifs reduces virus infectivity and leads to high-frequency deletion events (of one of the motifs) after infection: the resulting RNA species encode a wild type-like NC protein restoring full infectivity to the progeny virus particles. Additionally, the data suggest that this occurs only after infection. The deletion probably arises by intramolecular displacement of the replication complex between repeat sequences.     

Guanosine-5'-diphosphate at the 5' termini of reovirus RNA: evidence for a segmented genome within the virion

All ten double-stranded RNA fragments isolated from purified reovirus contain ppGp at the 5′ termini. The presence of a unique 5′-terminal nucleotide indicates that the viral genome in situ consists of segments which are synthesized as discrete units in infected cells. The penultimate base is a pyrimidine. This 5′ sequence, ppGpPyp, is identical to that reported previously for the ten reovirus messenger RNA species synthesized in vitro. The results indicate that the double-stranded RNA segments are perfect duplexes which are transcribed end-to-end by the virion-associated RNA polymerase.     

Interaction of small ribosomal and transfer RNAs with a protein from Leishmania donovani.

Using synthetic antisense RNA from the 5'-untranslated region of the beta-tubulin gene as probe in gel retardation assays, a heat stable RNA-binding factor was identified in promastigotes of the kinetoplastid protozoan Leishmania donovani. The same or similar factors interact with several small ribosomal RNA (srRNA) species and, more weakly, with tRNA, as shown by binding and competition experiments. Deletion analysis indicated involvement of repeated purine-rich motifs on the antisense RNA, in the reaction. Related, conserved motifs occur on at least two of the srRNAs. By a modified Western blot assay, the RNA-binding species was identified as a single, small polypeptide. The activity is apparently specific for the promastigote stage of the parasite, being undetectable in amastigotes. The properties of this RNA-binding factor suggest that it is a novel, previously uncharacterized protein.     

RNA structural motif recognition based on least-squares distance

RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.     

RNA structural motifs: building blocks of a modular biomolecule

RNAs are modular biomolecules, composed largely of conserved structural subunits, or motifs. These structural motifs comprise the secondary structure of RNA and are knit together via tertiary interactions into a compact, functional, three-dimensional structure and are to be distinguished from motifs defined by sequence or function. A relatively small number of structural motifs are found repeatedly in RNA hairpin and internal loops, and are observed to be composed of a limited number of common 'structural elements'. In addition to secondary and tertiary structure motifs, there are functional motifs specific for certain biological roles and binding motifs that serve to complex metals or other ligands. Research is continuing into the identification and classification of RNA structural motifs and is being initiated to predict motifs from sequence, to trace their phylogenetic relationships and to use them as building blocks in RNA engineering.     

Experimental RNomics: identification of 140 candidates for small non-messenger RNAs in the plant Arabidopsis thaliana

BACKGROUND: Genomes from all organisms known to date express two types of RNA molecules: messenger RNAs (mRNAs), which are translated into proteins, and non-messenger RNAs, which function at the RNA level and do not serve as templates for translation. RESULTS: We have generated a specialized cDNA library from Arabidopsis thaliana to investigate the population of small non-messenger RNAs (snmRNAs) sized 50-500 nt in a plant. From this library, we identified 140 candidates for novel snmRNAs and investigated their expression, abundance, and developmental regulation. Based on conserved sequence and structure motifs, 104 snmRNA species can be assigned to novel members of known classes of RNAs (designated Class I snmRNAs), namely, small nucleolar RNAs (snoRNAs), 7SL RNA, U snRNAs, as well as a tRNA-like RNA. For the first time, 39 novel members of H/ACA box snoRNAs could be identified in a plant species. Of the remaining 36 snmRNA candidates (designated Class II snmRNAs), no sequence or structure motifs were present that would enable an assignment to a known class of RNAs. These RNAs were classified based on their location on the A. thaliana genome. From these, 29 snmRNA species located to intergenic regions, 3 located to intronic sequences of protein coding genes, and 4 snmRNA candidates were derived from annotated open reading frames. Surprisingly, 15 of the Class II snmRNA candidates were shown to be tissue-specifically expressed, while 12 are encoded by the mitochondrial or chloroplast genome. CONCLUSIONS: Our study has identified 140 novel candidates for small non-messenger RNA species in the plant A. thaliana and thereby sets the stage for their functional analysis.     

Arrangement of 3D structural motifs in ribosomal RNA

Structural 3D motifs in RNA play an important role in the RNA stability and function. Previous studies have focused on the characterization and discovery of 3D motifs in RNA secondary and tertiary structures. However, statistical analyses of the distribution of 3D motifs along the RNA appear to be lacking. Herein, we present a novel strategy for evaluating the distribution of 3D motifs along the RNA chain and those motifs whose distributions are significantly non-random are identified. By applying it to the X-ray structure of the large ribosomal subunit from Haloarcula marismortui, helical motifs were found to cluster together along the chain and in the 3D structure, whereas the known tetraloops tend to be sequentially and spatially dispersed. That the distribution of key structural motifs such as tetraloops differ significantly from a random one suggests that our method could also be used to detect novel 3D motifs of any size in sufficiently long/large RNA structures. The motif distribution type can help in the prediction and design of 3D structures of large RNA molecules.     

Three unique viral RNA species of snowshoe hare and La Crosse bunyaviruses.

Two-dimensional gel electrophoreses of RNase T1-derived oligonucleotides of the three individual RNA segments of the bunyavirus snowshow hare virus indicate that its three RNA segments possess distinct nucleotide sequences. The fingerprints of the RNA species of snowshoe hare virus differ from those of the antigenically closely related La Crosse virus. Three viral RNA species have been identified in preparations of Melao and Trivittatus as well as snowshoe hare, Lumbo, and La Crosse bunyaviruses.