Stable Stem Loop Research Articles

Serology-Enabled Discovery of Genetically Diverse Hepaciviruses in a New Host

Genetic and biological characterization of new hepaciviruses infecting animals contributes to our understanding of the ultimate origins of hepatitis C virus (HCV) infection in humans and dramatically enhances our ability to study its pathogenesis using tractable animal models. Animal homologs of HCV include a recently discovered canine hepacivirus (CHV) and GB virus B (GBV-B), both viruses with largely undetermined natural host ranges. Here we used a versatile serology-based approach to determine the natural host of the only known nonprimate hepacivirus (NPHV), CHV, which is also the closest phylogenetic relative of HCV. Recombinant protein expressed from the helicase domain of CHV NS3 was used as antigen in the luciferase immunoprecipitation system (LIPS) assay to screen several nonprimate animal species. Thirty-six samples from 103 horses were immunoreactive, and viral genomic RNA was present in 8 of the 36 seropositive animals and none of the seronegative animals. Complete genome sequences of these 8 genetically diverse NPHVs showed 14% (range, 6.4% to 17.2%) nucleotide sequence divergence, with most changes occurring at synonymous sites. RNA secondary structure prediction of the 383-base 5' untranslated region of NPHV was refined and extended through mapping of polymorphic sites to unpaired regions or (semi)covariant pairings. Similar approaches were adopted to delineate extensive RNA secondary structures in the coding region of the genome, predicted to form 27 regularly spaced, thermodynamically stable stem-loops. Together, these findings suggest a promising new nonprimate animal model and provide a database that will aid creation of functional NPHV cDNA clones and other novel tools for hepacivirus studies.

Identification and characterization of the microRNAs and their targets in Salmo salar

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 18 to 26 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. The study resulted in novel 75 precursor miRNAs containing 102 mature sequences belonging to 46 families in an important aquatic environmental monitoring fish (Salmo salar). All the miRNA families (let-7, mir-1, 7, 9, 21, 22, 92, 96, 122, 126, 128, 129, 132, 133, 142, 144, 147, 148, 196, 202, 212, 223, 375, 429, 430, 449, 451, 457, 466, 682, 700, 1388, 1594, 1600, 1607, 1616, 1642, 1681, 1701, 1720, 1772, 1782, 1787, 1814, 2189 and 3540) are found for the first time in S. salar. All 75 miRNA precursors form stable minimum free energy stem loop and the mature miRNAs reside in the stem portion of the stem loop structure. Their target proteins are involved in transcription factors (28%), metabolism (23%), signaling (18%), transportation (9%), immunity (8%), stress related activity (5%), cancer and tumor related activity (5%), growth and development (3%), and cell division (1%).

The novel 172 sheep (Ovis aries) microRNAs and their targets

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about ≈22 nucleotides in length. The comparative genomics approach due to their conserved nature is a good source for the novel miRNAs discovery. In this study, total 172 novel miRNAs from 140 precursor sequences belonging to 114 families were identified in sheep (Ovis aries), the most important livestock animal. All the miRNA families (oar-mir-95, 129, 130, 186, 214, 219, 223, 324, 339, 423, 450, 499, 544, 562, 568, 584, 669, 671, 763, 935, 1281, 1282, 1306, 1552, 1584, 1587, 1603, 1607, 1706, 1711, 1718, 1732, 1738, 1761, 1771, 1777, 1778, 1780, 1807, 1843, 1895, 1930, 2127, 2139, 2182, 2284, 2287, 2295, 2296, 2310, 2311, 2314, 2315, 2316, 2320, 2359, 2378, 2381, 2382, 2395, 2400, 2404, 2410, 2412, 2423, 2426, 2435, 2470, 2477, 2482, 2487, 2881, 2883, 2885, 2888, 2889, 2896, 2901, 2904, 2917, 2964, 3063, 3064, 3074, 3080, 3432, 3529, 3533, 3613, 3649, 3654, 3658, 3661, 3662, 3940, 3960, 4273, 4426, 4447, 4459, 4468, 4493, 4507, 4647, 4680, 4785, 4788, 4800, 5102, 5105, 5109, 5115, 5125 and 5132) are found for the first time in Sheep. All 140 miRNA precursors form stable minimum free energy stem loop and the mature miRNAs reside in the stem portion of the stem loop structure. Their putative targets are involved in transcription factors (26%), signaling (19%), metabolism (18%), transportation (10%), immunity (9%), cancer and tumor related (5%), growth and development (5%), stress related (4%), and structural proteins (3%).

Mitochondrial genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis

We determined the complete mitochondrial (mt) genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis. The mt genomes of A. a. coreae and A. a. chejuensis are 16,260 and 16,261 base pairs in length, respectively. The general features of the 13 protein-coding genes of the two species are similar to those of other rodents. The TAG termination codon for NADH dehydrogenase subunit (ND) 3 is unique to Apodemus in the Muroidea. The L-strand replication origin has the potential to form a stable stem–loop structure. Within the control region, a termination-associated sequence and several conserved sequence blocks were observed. The diversity of the 13 protein-coding genes, 2 rRNAs, and 1 control region between the two species ranged between 0.005 (ATP8) and 0.027 (ND4L).

The mitochondrial genome of Apodemus peninsulae (Rodentia, Muridae)

The complete mitochondrial (mt) genome of the Korean field mouse Apodemus peninsulae was sequenced and found to be 16,266 bp in length. The mt protein-coding genes of A. peninsulae had ATG, GTG, ATC, and ATA as initiation codons and TAA, TAG, TA, and T as termination codons. Two forms each of trnL and trnS and the three tRNA clusters, IQM, WANCY, and HSL were identified, as in the typical Rodentia mt genome. Among tRNAs, abnormal cloverleaf structure of trnS(AGY) was identified in DHU arm. The l-strand replication origin has the potential to form a stable stem–loop structure and control region has several conserved sequence elements.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Identification of microRNAs in ecological model plant <i>Mimulus</i>

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 20 to 24 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. This study resulted in 34 miRNAs belonging to 21 families in ecological model plant Mimulus. All the miRNA families (mir 156, 157, 159, 160, 164, 172, 319, 393, 395, 397, 399, 400, 403, 417, 419, 472, 782, 854, 858, 867 and 2112) are found for the first time in Mimulus. The MIR-399 is found as precursor miRNA cluster with 4 mature sequences. All 34 miRNA precursors form stable minimum free energy stem loop structure as their orthologues form and the mature miRNAs reside in the stem portion of the stem loop structure. Twenty eight are from Mimulus guttatus and six miRNAs belong to Mimulus lewisii. Their targets consist of dihy-droflavonol-4-reductase, cycloidea-like protein, DNA-directed RNA polymerase II, maturase (matR) and transcription factors like; squamosa-promoter binding, MYB, palmate-like pentafoliata 1.

MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

Functional Analysis of RNA Structures Present at the 3′ Extremity of the Murine Norovirus Genome: the Variable Polypyrimidine Tract Plays a Role in Viral Virulence

Interactions of host cell factors with RNA sequences and structures in the genomes of positive-strand RNA viruses play various roles in the life cycles of these viruses. Our understanding of the functional RNA elements present in norovirus genomes to date has been limited largely to in vitro analysis. However, we recently used reverse genetics to identify evolutionarily conserved RNA structures and sequences required for norovirus replication. We have now undertaken a more detailed analysis of RNA structures present at the 3' extremity of the murine norovirus (MNV) genome. Biochemical data indicate the presence of three stable stem-loops, including two in the untranslated region, and a single-stranded polypyrimidine tract [p(Y)] of variable length between MNV isolates, within the terminal stem-loop structure. The well-characterized host cell pyrimidine binding proteins PTB and PCBP bound the 3'-untranslated region via an interaction with this variable sequence. Viruses lacking the p(Y) tract were viable both in cell culture and upon mouse infection, demonstrating that this interaction was not essential for virus replication. However, competition analysis with wild-type MNV in cell culture indicated that the loss of the p(Y) tract was associated with a fitness cost. Furthermore, a p(Y)-deleted mutant showed a reduction in virulence in the STAT1(-/-) mouse model, highlighting the role of RNA structures in norovirus pathogenesis. This work highlights how, like with other positive-strand RNA viruses, RNA structures present at the termini of the norovirus genome play important roles in virus replication and virulence.

Three discontinuous loop nucleotides in the 3′ terminal stem-loop are required for Red clover necrotic mosaic virus RNA-2 replication

The genome of Red clover necrotic mosaic virus (RCNMV) consists of positive-sense, single-stranded RNA-1 and RNA-2. The 29 nucleotides at the 3' termini of both RNAs are nearly identical and are predicted to form a stable stem-loop (SL) structure, which is required for RCNMV RNA replication. Here we performed a systematic mutagenesis of the RNA-2 3' SL to identify the nucleotides critical for replication. Infectivity and RNA replication assays indicated that the secondary structure of the 3' SL and its loop sequence UAUAA were required for RNA replication. Single-nucleotide substitution analyses of the loop further pinpointed three discontinuous nucleotides (L1U, L2A, and L4A) that were vital for RNA replication. A 3-D model of the 3' SL predicted the existence of a pocket formed by these three nucleotides that could be involved in RNA-protein interaction. The functional groups of the bases participating in this interaction at these positions are discussed.

A study on genomic distribution and sequence features of human long inverted repeats reveals species‐specific intronic inverted repeats

The inverted repeats present in a genome play dual roles. They can induce genomic instability and, on the other hand, regulate gene expression. In the present study, we report the distribution and sequence features of recombinogenic long inverted repeats (LIRs) that are capable of forming stable stem-loops or palindromes within the human genome. A total of 2551 LIRs were identified, and 37% of them were located in long introns (largely > 10 kb) of genes. Their distribution appears to be random in introns and is not restrictive, even for regions near intron-exon boundaries. Almost half of them comprise TG/CA-rich repeats, inversely arranged Alu repeats and MADE1 mariners. The remaining LIRs are mostly unique in their sequence features. Comparative studies of human, chimpanzee, rhesus monkey and mouse orthologous genes reveal that human genes have more recombinogenic LIRs than other orthologs, and over 80% are human-specific. The human genes associated with the human-specific LIRs are involved in the pathways of cell communication, development and the nervous system, as based on significantly over-represented Gene Ontology terms. The functional pathways related to the development and functions of the nervous system are not enriched in chimpanzee and mouse orthologs. The findings of the present study provide insight into the role of intronic LIRs in gene regulation and primate speciation.

Intergenic regions of Borrelia plasmids contain phylogenetically conserved RNA secondary structure motifs

BackgroundBorrelia species are unusual in that they contain a large number of linear and circular plasmids. Many of these plasmids have long intergenic regions. These regions have many fragmented genes, repeated sequences and appear to be in a state of flux, but they may serve as reservoirs for evolutionary change and/or maintain stable motifs such as small RNA genes.ResultsIn an in silico study, intergenic regions of Borrelia plasmids were scanned for phylogenetically conserved stem loop structures that may represent functional units at the RNA level. Five repeat sequences were found that could fold into stable RNA-type stem loop structures, three of which are closely linked to protein genes, one of which is a member of the Borrelia lipoprotein_1 super family genes and another is the complement regulator-acquiring surface protein_1 (CRASP-1) family. Modeled secondary structures of repeat sequences display numerous base-pair compensatory changes in stem regions, including C-G→A-U transversions when orthologous sequences are compared. Base-pair compensatory changes constitute strong evidence for phylogenetic conservation of secondary structure.ConclusionIntergenic regions of Borrelia species carry evolutionarily stable RNA secondary structure motifs. Of major interest is that some motifs are associated with protein genes that show large sequence variability. The cell may conserve these RNA motifs whereas allow a large flux in amino acid sequence, possibly to create new virulence factors but with associated RNA motifs intact.

The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop.

Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem-loops and a conserved long-range interaction between heptanucleotide sequences 5'-CCCUGUC-3' in R/U5 and 5'-GACAGGG-3' in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5' UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

Transactivation-response element (TAR) is a stable stem-loop structure of HIV RNA, which plays a crucial role during the life cycle of the virus. The apical loop of TAR acts as a binding site for essential cellular cofactors required for the replication of HIV. High-affinity aptamers directed against the apical loop of TAR have been identified by the SELEX approach. The RNA aptamers with the highest affinity for TAR fold as hairpins and form kissing complexes with the targeted RNA through loop-loop interactions. The aptamers with the strongest binding properties all possess a GA base pair combination at the loop-closing position. Using liquid-crystal NMR methodology, we have obtained a structural model in solution of a TAR-aptamer kissing complex with an unprecedented accuracy. This high-resolution structure reveals that the GA base pair is unilaterally shifted toward the 5' strand and is stabilized by a network of intersugar hydrogen bonds. This specific conformation of the GA base pair allows for the formation of two supplementary stable base-pair interactions. By systematic permutations of the loop-closing base pair, we establish that the identified atomic interactions, which form the basis for the high stability of the complex, are maintained in several other kissing complexes. This study rationalizes the stabilizing role of the loop-closing GA base pairs in kissing complexes and may help the development or improvement of drugs against RNA loops of viruses or pathogens as well as the conception of biochemical tools targeting RNA hairpins involved in important biological functions.

Structure of Yeast Dom34: A PROTEIN RELATED TO TRANSLATION TERMINATION FACTOR Erf1 AND INVOLVED IN No-Go DECAY

The yeast protein Dom34 has been described to play a critical role in a newly identified mRNA decay pathway called No-Go decay. This pathway clears cells from mRNAs inducing translational stalls through endonucleolytic cleavage. Dom34 is related to the translation termination factor eRF1 and physically interacts with Hbs1, which is itself related to eRF3. We have solved the 2.5-A resolution crystal structure of Saccharomyces cerevisiae Dom34. This protein is organized in three domains with the central and C-terminal domains structurally homologous to those from eRF1. The N-terminal domain of Dom34 is different from eRF1. It adopts a Sm-fold that is often involved in the recognition of mRNA stem loops or in the recruitment of mRNA degradation machinery. The comparison of eRF1 and Dom34 domains proposed to interact directly with eRF3 and Hbs1, respectively, highlights striking structural similarities with eRF1 motifs identified to be crucial for the binding to eRF3. In addition, as observed for eRF1 that enhances eRF3 binding to GTP, the interaction of Dom34 with Hbs1 results in an increase in the affinity constant of Hbs1 for GTP but not GDP. Taken together, these results emphasize that eukaryotic cells have evolved two structurally related complexes able to interact with ribosomes either paused at a stop codon or stalled in translation by the presence of a stable stem loop and to trigger ribosome release by catalyzing chemical bond hydrolysis.

MRNA secondary structure modulates the translation of organophosphate hydrolase (OPH) in E. coli

Organophosphate hydrolases (OPHs), involved in hydrolytic cleavage of structurally diverse organophosphates are coded by a plasmid borne, highly conserved organophosphate degrading (opd) gene. An inverted repeat sequence found in the signal coding region of the opd gene was found to be responsible for inducing a stable stem loop structure with a DeltaG of -23.1 kcal/mol. This stem loop structure has shown significant influence on the expression levels of organophosphate hydrolase (OPH) in E. coli. When the signal coding region comprising the inverted repeat sequence was deleted a approximately 3.28 fold increase in the expression levels of OPH was noticed in E. coli BL21 cells. Mutations in the inverted repeat region, especially at the third position of the codon, to a non-complementary base destabilized the secondary structure of opd mRNA. When such opd variant, opd' was expressed, the expression levels were found to be similar to expression levels coded by the construct generated by deleting the signal peptide coding region. Deletion of signal peptide did not influence the folding and activity of OPH. Though high level induction has resulted in accumulation of OPH as inclusion bodies, modulation of expression levels by reducing the copy number of the expression plasmid, inducer concentration and growth temperature has produced majority of the protein in soluble and active form.

Programmed Ribosomal Frameshifting in SIV Is Induced by a Highly Structured RNA Stem–Loop

Simian immunodeficiency virus (SIV), like its human homologues (HIV-1, HIV-2), requires a -1 translational frameshift event to properly synthesize all of the proteins required for viral replication. The frameshift mechanism is dependent upon a seven-nucleotide slippery sequence and a downstream RNA structure. In SIV, the downstream RNA structure has been proposed to be either a stem–loop or a pseudoknot. Here, we report the functional, structural and thermodynamic characterization of the SIV frameshift site RNA. Translational frameshift assays indicate that a stem–loop structure is sufficient to promote efficient frameshifting in vitro. NMR and thermodynamic studies of SIV RNA constructs of varying length further support the absence of any pseudoknot interaction and indicate the presence of a stable stem–loop structure. We determined the structure of the SIV frameshift-inducing RNA by NMR. The structure reveals a highly ordered 12 nucleotide loop containing a sheared G-A pair, cross-strand adenine stacking, two G-C base-pairs, and a novel CCC triloop turn. The loop structure and its high thermostability preclude pseudoknot formation. Sequence conservation and modeling studies suggest that HIV-2 RNA forms the same structure. We conclude that, like the main sub-groups of HIV-1, SIV and HIV-2 utilize stable stem–loop structures to function as a thermodynamic barrier to translation, thereby inducing ribosomal pausing and frameshifting.

RNA channelling by the archaeal exosome

Exosomes are complexes containing 3' --> 5' exoribonucleases that have important roles in processing, decay and quality control of various RNA molecules. Archaeal exosomes consist of a hexameric core of three active RNase PH subunits (ribosomal RNA processing factor (Rrp)41) and three inactive RNase PH subunits (Rrp42). A trimeric ring of subunits with putative RNA-binding domains (Rrp4/cep1 synthetic lethality (Csl)4) is positioned on top of the hexamer on the opposite side to the RNA degrading sites. Here, we present the 1.6 A resolution crystal structure of the nine-subunit exosome of Sulfolobus solfataricus and the 2.3 A structure of this complex bound to an RNA substrate designed to be partly trimmed rather than completely degraded. The RNA binds both at the active site on one side of the molecule and on the opposite side in the narrowest constriction of the central channel. Multiple substrate-binding sites and the entrapment of the substrate in the central channel provide a rationale for the processive degradation of extended RNAs and the stalling of structured RNAs.

Betaine, Dimethyl Sulfoxide, and 7-Deaza-dGTP, a Powerful Mixture for Amplification of GC-Rich DNA Sequences

Currently, polymerase chain reaction is the most used technique in many laboratories for either diagnostic or molecular biology purposes. Despite the large number of DNA sequences that can be easily analyzed, some GC-rich sequences are refractory to amplification due to the formation of secondary intramolecular structures. To overcome this problem, several molecules have been described to improve polymerization. Here we show that a combination of three additives--betaine, dimethyl sulfoxide, and 7-deaza-dGTP--was essential to achieve amplification of DNA sequences of three disease genes showing a GC content ranging from 67 to 79%.

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex.