A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication.

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.     

Facile means for quantifying microRNA expression by real-time PCR

MicroRNAs (miRNAs) are 20-24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having asfew as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.     

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.     

Diversity and evolution of mitochondrial RNA editing systems

'RNA editing' describes the programmed alteration of the nucleotide sequence of an RNA species, relative to the sequence of the encoding DNA. The phenomenon encompasses two generic patterns of nucleotide change, 'insertion/deletion' and 'substitution', defined on the basis of whether the sequence of the edited RNA is colinear with the DNA sequence that encodes it. RNA editing is mediated by a variety of pathways that are mechanistically and evolutionarily unrelated. Messenger, ribosomal, transfer and viral RNAs all undergo editing in different systems, but well-documented cases of this phenomenon have so far been described only in eukaryotes, and most often in mitochondria. Editing of mRNA changes the identity of encoded amino acids and may create translation initiation and termination codons. The existence of RNA editing violates one of the long-accepted tenets of genetic information flow, namely, that the amino acid sequence of a protein can be directly predicted from the corresponding gene sequence. Particular RNA editing systems display a narrow phylogenetic distribution, which argues that such systems are derived within specific eukaryotic lineages, rather than representing traits that ultimately trace to a common ancestor of eukaryotes, or even further back in evolution. The derived nature of RNA editing raises intriguing questions about how and why RNA editing systems arise, and how they become fixed as additional, essential steps in genetic information transfer.     

Advantages and disadvantages of using PCR techniques to characterize transgenic plants

The polymerase chain reaction (PCR) revolutionized molecular biology to a similar extent as the discovery of plasmids and restriction endonucleases. However, there are some limitations to the use of PCR. Transgenic plants containing potato spindle tuber viroid (PSTVd) cDNA constructs, demonstrated to become de novo methylated upon PSTVd infection, represent a good example to illustrate the advantages of PCR. PSTVd is a 359 nt long autonomously replicating plant pathogenic RNA where all of its enzymatic requirements are entirely provided by the host cell. In addition, viroids that propagate without a DNA intermediate barely tolerate nucleotide substitutions of their RNA genome without losing infectivity. PCR is the method of choice to characterize the sequence context of genome-integrated viroid cDNA or of reverse transcribed PSTVd RNA, and can hardly be replaced by any alternative procedure. Furthermore, the precise examination of DNA methylation patterns (genomic sequencing) is entirely dependent on PCR. In contrast, the use of PCR is critical for the determination of copy number and arrangement of transgene constructs. Here, the advantages and disadvantages of PCR are discussed and protocols for PCR amplification of cDNA, genomic DNA, and bisulfite-treated DNA from transgenic plants are presented.     

The phenomenon of RNA interference and development of organism

RNA interference consists in specific mRNA degradation in response to introduction of a double-stranded RNA, homologous in nucleotide sequence. RNA interference was found in eukaryotes and is used in genomics as a powerful method to determine the functions of genes with known nucleotide sequences. RNA interference is considered as a tool of protection against viruses and harmful consequences of mobile elements' transposals. The involvement of the components of RNA interference is considered in spermatogenesis of Drosophila melanogaster and regulation of the expression of genes in Caenorhabditis elegans responsible for temporal patterns of development. The role of RNQA interference in stem cell formation and functioning is also considered.     

Prediction of interacting single-stranded RNA bases by protein-binding patterns

Prediction of protein-RNA interactions at the atomic level of detail is crucial for our ability to understand and interfere with processes such as gene expression and regulation. Here, we investigate protein binding pockets that accommodate extruded nucleotides not involved in RNA base pairing. We observed that most of the protein-interacting nucleotides are part of a consecutive fragment of at least two nucleotides whose rings have significant interactions with the protein. Many of these share the same protein binding cavity and more than 30% of such pairs are π-stacked. Since these local geometries cannot be inferred from the nucleotide identities, we present a novel framework for their prediction from the properties of protein binding sites.First, we present a classification of known RNA nucleotide and dinucleotide protein binding sites and identify the common types of shared 3-D physicochemical binding patterns. These are recognized by a new classification methodology that is based on spatial multiple alignment. The shared patterns reveal novel similarities between dinucleotide binding sites of proteins with different overall sequences, folds and functions. Given a protein structure, we use these patterns for the prediction of its RNA dinucleotide binding sites. Based on the binding modes of these nucleotides, we further predict an RNA fragment that interacts with those protein binding sites. With these knowledge-based predictions, we construct an RNA fragment that can have a previously unknown sequence and structure. In addition, we provide a drug design application in which the database of all known small-molecule binding sites is searched for regions similar to nucleotide and dinucleotide binding patterns, suggesting new fragments and scaffolds that can target them.     

Nucleotide sequence at the junction between the nonstructural and the structural genes of the semliki forest virus genome.

The nucleotide sequence at the junction between the nonstructural and the structural genes of the Semliki Forest virus 42S RNA genome has been determined from cloned cDNA. With the aid of S1-mapping, we have located the 5' end of the viral 26S RNA on this sequence. The 26S RNA is homologous to the 3' end of the 42S RNA and is used as a messenger for the structural proteins of the virus. The nucleotide sequence in the noncoding 5' region of the 26S RNA (51 bases) was thus established, completing the primary structure of the 26S RNA molecule (for earlier sequence work, see Garoff et al., Proc. Natl. Acad. Sci. U.S.A. 77:6376-6380, 1980, and Garoff et al., Nature (London) 288:236-241, 1980). An examination of the nucleotide sequences upstream from the initiator codon for the structural proteins on the 42S RNA genome shows that all reading frames are effectively blocked by stop codons, which means that the nonstructural genes in the 5' end of the 42S RNA molecule do not overlap with the structural ones at the 3' end of the molecule.     

RNA Interference and Development

RNA interference consists in specific mRNA degradation in response to introduction of a double-stranded RNA, homologous in nucleotide sequence. RNA interference was found in eukaryotes and is used in genomics as a powerful method to determine the functions of genes with known nucleotide sequences. RNA interference is considered as a tool of protection against viruses and harmful consequences of mobile elements' transposals. The involvement of the components of RNA interference is considered in spermatogenesis of Drosophila melanogaster and regulation of the expression of genes in Caenorhabditis elegans responsible for temporal patterns of development. The role of RNA interference in stem cell formation and functioning is also considered.     

The prediction of virus mutation using neural networks and rough set techniques

Viral evolution remains to be a main obstacle in the effectiveness of antiviral treatments. The ability to predict this evolution will help in the early detection of drug-resistant strains and will potentially facilitate the design of more efficient antiviral treatments. Various tools has been utilized in genome studies to achieve this goal. One of these tools is machine learning, which facilitates the study of structure-activity relationships, secondary and tertiary structure evolution prediction, and sequence error correction. This work proposes a novel machine learning technique for the prediction of the possible point mutations that appear on alignments of primary RNA sequence structure. It predicts the genotype of each nucleotide in the RNA sequence, and proves that a nucleotide in an RNA sequence changes based on the other nucleotides in the sequence. Neural networks technique is utilized in order to predict new strains, then a rough set theory based algorithm is introduced to extract these point mutation patterns. This algorithm is applied on a number of aligned RNA isolates time-series species of the Newcastle virus. Two different data sets from two sources are used in the validation of these techniques. The results show that the accuracy of this technique in predicting the nucleotides in the new generation is as high as 75 %. The mutation rules are visualized for the analysis of the correlation between different nucleotides in the same RNA sequence.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

Molecular genetic analysis of the two morphs of sea star shrimp <Emphasis Type="Italic">Periclimemes soror</Emphasis> Nobili, 1904, the symbionts of tropic sea stars

Two morphologically similar morphs of the Periclimemes soror shrimps were subjected to nucleotide sequence analysis of the mitochondrial COI gene fragment and highly variable nuclear fragment, D1–D2 domain of the large subunit of their ribosomal RNA genes. These shrimps are widely distributed in the Indo-West-Pacific and are obligate symbionts of sea stars. The morphs are different in color patterns, as well as in specific reaction relative to their hosts. The nucleotide sequence data obtained revealed no differentiation relative to the fragments examined.     

Nucleotide sequence complexities, molecular weights, and poly(A) content of the vesicular stomatitis virus mRNA species.

Poly(A)-containing vesicular stomatitis virus mRNA species synthesized in vesicular stomatitis virus-infected cells have been separated into four bands by electrophoresis on formamide-polyacrylamide gels. Two-dimensional fingerprints of ribonuclease T-1 and ribonuclease A digests of the RNA from each band show that they contain unique oligonucleotide sequences as well as 60 to 125 nucleotides of poly(A). The fingerprints were used to determine the nucleotide sequence complexities of RNA from three of the bands. Two contain nucleotide sequences which account completely for their molecular weights (0.70 times 10-6 and 0.55 times 10-6) determined by gel electrophoresis and sedimentation rate, and, therefore, these are radiochemically pure RNA species. The most rapidly migrating band must contain two ro three different RNA species since it has a molecular weight of 0.28 times 10-6, determined by physical methods, and a nucleotide sequence complexity two to three times that expected for a pure RNA species of this size. These data are in complete accord with translational studies (accompanying paper) which show that each of the two pure RNA species codes for a distinct viral protein, whereas the third codes for two viral proteins. From the molecular weight and sequence complexity determinations on mRNA from the bands, we conclude that most of the vesicular stomatitis virus genome is transcribed into discrete mRNA species.     

Primary and secondary structure of 7-3 (K) RNA of Novikoff hepatoma

7-3 RNA (also known as K-RNA and 7SK-RNA) is a distinct small RNA found in insect to mammalian cells. Previous studies showed that this RNA is not capped, contains no modified nucleotides, is conserved through evolution, is synthesized by RNA polymerase III, and, in part, is associated by polyribosomes. In this study, the complete nucleotide sequence of 7-3 RNA was determined by RNA-sequencing methods, and the sequence is compared with several small RNAs and repetitive DNA sequences for homology. This 330-nucleotide-long RNA contained pppGp as its 5' terminus and exhibited heterogeneity with respect to the 3'-terminal AoH. The nucleotide sequence is: (sequence in text) The RNA is G-C rich, and evidence is presented that 7-3 RNA is in a ribonucleoprotein particle in the cytoplasm.     

Recurrent RNA motifs as probes for studying RNA-protein interactions in the ribosome

To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.