Identification of potential conserved RNA secondary structure throughout influenza A coding regions

Influenza A is a negative sense RNA virus of significant public health concern. While much is understood about the life cycle of the virus, knowledge of RNA secondary structure in influenza A virus is sparse. Predictions of RNA secondary structure can focus experimental efforts. The present study analyzes coding regions of the eight viral genome segments in both the (+) and (-) sense RNA for conserved secondary structure. The predictions are based on identifying regions of unusual thermodynamic stabilities and are correlated with studies of suppression of synonymous codon usage (SSCU). The results indicate that secondary structure is favored in the (+) sense influenza RNA. Twenty regions with putative conserved RNA structure have been identified, including two previously described structured regions. Of these predictions, eight have high thermodynamic stability and SSCU, with five of these corresponding to current annotations (e.g., splice sites), while the remaining 12 are predicted by the thermodynamics alone. Secondary structures with high conservation of base-pairing are proposed within the five regions having known function. A combination of thermodynamics, amino acid and nucleotide sequence comparisons along with SSCU was essential for revealing potential secondary structures.     

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

It is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.     

Predicted RNA secondary structures for the conserved regions in dengue virus

Dengue fever, dengue hemorrhagic fever and dengue shock syndrome are the prevalent mosquito borne viral infections worldwide. The dengue virus belongs to the genus flavivirus with conserved RNA domains peptidase_S7 and dexHc among its members. The secondary structures for RNA domains peptidase_S7 and DexHc are hence predicted and discussed with other known viral RNA structures to glean structural insights through comparison.     

Protein-coding structured RNAs: A computational survey of conserved RNA secondary structures overlapping coding regions in drosophilids

Functional RNA elements can be embedded also within exonic sequences coding for functional proteins. While not uncommon in viruses, only a few examples of this type have been described in some detail for eukaryotic genomes. Here we use RNAz and RNAcode, two comparative genomics methods that measure signatures of stabilizing selection acting on RNA secondary structure and peptide sequence, resp., to survey the fruit fly genomes. We estimate that there might be on the order of 1000 loci that are subject to dual selection pressure. The used genome-wide screens also expose the limitations of the currently available methods.     

Statistical method for predicting protein coding regions in nucleic acid sequences

Protein coding regions of a genome fragment can be mathematicallypredicted by studying variations in the statistical propertiesor by searching the signals characteristic of the junctionsbetween the coding and non-coding regions. We propose here anew statistical method using correspondence analysis. This methoddoes not use any reference codon set but takes into accountthe codon usage homogeneity along the studied genome fragment.Comparison with previously published methods especially the‘codon usage method’ of Staden has been made, andtwo examples are presented here. Applications to analysis ofprokaryotic operon and eukaryotic split genes are also discussed.Use of the method has also shown two structures not previouslydescribed: i) in the human prt gene, a strong triplet structureexists in a non-coding region; ii) in the human tp-a codon usageis not uniform between the different exons Received on September 25, 1986     

Influence of 5' proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors

The effects of 5' proximal secondary structure in mRNA molecules on their translation and on their interaction with the eukaryotic initiation factors (eIF)-4F, eIF-4A, and eIF-4B have been examined. Secondary structures were generated in the 5' noncoding region of rabbit globin and reovirus mRNAs by means of hybridization with cDNA molecules. cDNAs hybridized to the first 15 bases downstream from the cap inhibited the translation of the mRNAs in both reticulocyte and wheat germ lysates. The degree of inhibition was directly related to the monovalent ion concentration and inversely related to reaction temperature. These hybrid structures also reduced the competitive ability of the messages. Hybrid structures beginning downstream from the first 15 bases did not inhibit the translation of beta-globin mRNA or reovirus s3 mRNA. None of the hybrid structures were detrimental to the interaction of the mRNAs with the 26-kDa cap binding protein of eIF-4F, as determined by chemical cross-linking assays. However, in the presence of ATP, hybrid structures immediately adjacent to the cap severely inhibited the cross-linking to the p46 subunit of eIF-4F or to additional eIF-4A or eIF-4B. In order to account for these observations, a two-step mechanism is proposed for the interaction of eIF-4F with the 5' end of an mRNA molecule. The first step involves a weak initial interaction of the p26 subunit with the cap. The second step requires the hydrolysis of ATP and results in the formation of a stable initiation factor-mRNA complex, which may involve eIF-4A and eIF-4B. This second step is inhibited by the presence of 5' proximal secondary structure. In any event, our results demonstrate that the effect of mRNA structure on translation rate depends strongly on its position with respect to the 5' end and that this effect is due at least in part to an inhibition of the action of initiation factors normally required for the unwinding of structure.     

Data mining of imperfect double-stranded RNA in 3' untranslated regions of eukaryotic mRNAs

Recent developments in the study of RNA silencing indicate that double-stranded RNA (dsRNA) can be used in eukaryotes to block expression of a corresponding cellular gene. There is also a large class of small non-coding RNAs having potential to form a distinct, stable stem-loop in numbers of eukaryotic genomes. We had reported that a large imperfect dsRNA structure with hundreds of base-pairs (bp) in the 3' untranslated region (3' UTR) of cytotoxic ribonuclease was correlated with the translation suppression. In this study, we search for such dsRNAs in a 3' UTR database. The occurrence rate of large dsRNA in 3' UTRs ranges from 0.01% in plant to 0.30% in vertebrate mRNAs. However, small imperfect dsRNAs of ~ 30 bp are much more prevalent than large ones. The small dsRNAs are statistically very significant and uniquely well-ordered. Most of them have the conserved structural features of pre-miRNAs. Our data mining of the dsRNAs in the 3' UTR database can be used to explore RNA-based regulation of gene expression.     

Occurrence of protein structure elements in conserved sequence regions

Background  

Conserved protein sequence regions are extremely useful for identifying and studying functionally and structurally important regions. By means of an integrated analysis of large-scale protein structure and sequence data, structural features of conserved protein sequence regions were identified.     

Determination of eukaryotic protein coding regions using neural networks and information theory.

Our previous work applied neural network techniques to the problem of discriminating open reading frame (ORF) sequences taken from introns versus exons. The method counted the codon frequencies in an ORF of a specified length, and then used this codon frequency representation of DNA fragments to train a neural net (essentially a Perceptron with a sigmoidal, or "soft step function", output) to perform this discrimination. After training, the network was then applied to a disjoint "predict" set of data to assess accuracy. The resulting accuracy in our previous work was 98.4%, exceeding accuracies reported in the literature at that time for other algorithms. Here, we report even higher accuracies stemming from calculations of mutual information (a correlation measure) of spatially separated codons in exons, and in introns. Significant mutual information exists in exons, but not in introns, between adjacent codons. This suggests that dicodon frequencies of adjacent codons are important for intron/exon discrimination. We report that accuracies obtained using a neural net trained on the frequency of dicodons is significantly higher at smaller fragment lengths than even our original results using codon frequencies, which were already higher than simple statistical methods that also used codon frequencies. We also report accuracies obtained from including codon and dicodon statistics in all six reading frames, i.e. the three frames on the original and complement strand. Inclusion of six-frame statistics increases the accuracy still further. We also compare these neural net results to a Bayesian statistical prediction method that assumes independent codon frequencies in each position. The performance of the Bayesian scheme is poorer than any of the neural based schemes, however many methods reported in the literature either explicitly, or implicitly, use this method. Specifically, Bayesian prediction schemes based on codon frequencies achieve 90.9% accuracy on 90 codon ORFs, while our best neural net scheme reaches 99.4% accuracy on 60 codon ORFs. "Accuracy" is defined as the average of the exon and intron sensitivities. Achievement of sufficiently high accuracies on short fragment lengths can be useful in providing a computational means of finding coding regions in unannotated DNA sequences such as those arising from the mega-base sequencing efforts of the Human Genome Project. We caution that the high accuracies reported here do not represent a complete solution to the problem of identifying exons in "raw" base sequences. The accuracies are considerably lower from exons of small length, although still higher than accuracies reported in the literature for other methods. Short exon lengths are not uncommon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond

Single-stranded regions in RNA secondary structure are important for RNA–RNA and RNA–protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals ‘well-determined’ single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit β-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA–RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.     

UTRdb: a specialized database of 5'- and 3'-untranslated regions of eukaryotic mRNAs.

The important role the untranslated regions of eukaryotic mRNAs may play in gene regulation and expression is now widely acknowledged. For this reason we developed UTRdb, a specialized database of 5'- and 3'-untranslated sequences of eukaryotic mRNAs cleaned from redundancy. UTRdb entries are enriched with specialized information not present in the primary databases, including the presence of functional patterns already demonstrated by experimental analysis to have some functional role. A collection of such patterns is being collected in UTRsite database (http://bio-www.ba.cnr.it:8000/srs5/) which can also be used with appropriate computational tools to detect known functional patterns contained in mRNA untranslated regions.     

Comparative sequence analysis as a tool for studying the secondary structure of mRNAs.

Analysis of phylogenetically conserved secondary structure has been important in the development of models for the secondary structure of structural RNAs. In this paper, we apply this type of analysis to several families of informational RNAs to evaluate its usefulness in developing secondary structure models for mRNAs and mRNA precursors. We observed many conserved helices in all mRNA groups analyzed. Three criteria were used to identify potential helices which were not conserved solely because of coding sequence constraints, and may therefore be important for the structure and function of the RNA. These results suggest that this approach will be useful in deriving secondary structure models for informational RNAs when used in conjunction with other complementary techniques, and in designing experiments to determine the functional significance of conserved base pairing interactions.     

Strong sequence patterns in eukaryotic promoter regions: Potential implications for DNA structure

• 1.1. Analysis of eukaryotic sequences reveals recurring trends in upstream regions. Oligomers composed of (G/C)_n and (A/T)_m blocks are preferentially flanked by (G/C)₂ doublets on their 3' rather than on their 5′ ends, that is (G/C)_nä(A/T)_m(G/C)₂ > (G/C)_n+2(A/T)_m.
• 2.2. These trends are stronger for larger n and smaller m. Additional trends are outlined below.
• 3.3. The trends are correlated with DNA structural parameters, in particular with twist and roll angles.
• 4.4. Generally, the trends hold if the base pair step joining the 5′ (G/C)₂ doublet to the (G/C)_n (A/T)_m oligomer is not undertwisted and is not strongly rolled into the major groove.
• 5.5. Other DNA parameters crucial for DNA-protein interactions are discussed as well.