Stable Stem Loop Research Articles

Evidence of Copper Nanoparticles and Poly I:C Modulating Cas9 Interaction and Cleavage of COR (Conserved Omicron RNA).

Conserved omicron RNA (COR) is a 40 base long 99.9% conserved sequence in SARS-CoV-2 Omicron variant, predicted to form a stable stem loop, the targeted cleavage of which can be an ideal next step in controlling the spread of variants. The Cas9 enzyme has been traditionally utilized for gene editing and DNA cleavage. Previously Cas9 has been shown to be capable of RNA editing under certain conditions. Here we investigated the ability of Cas9 to bind to single-stranded conserved omicron RNA (COR) and examined the effect of copper nanoparticles (Cu NPs) and/or polyinosinic-polycytidilic acid (poly I:C) on the RNA cleavage ability of Cas9. The interaction of the Cas9 enzyme and COR with Cu NPs was shown by dynamic light scattering (DLS) and zeta potential measurements and was confirmed by two-dimensional fluorescence difference spectroscopy (2-D FDS). The interaction with and enhanced cleavage of COR by Cas9 in the presence of Cu NPs and poly I:C was shown by agarose gel electrophoresis. These data suggest that Cas9-mediated RNA cleavage may be potentiated at the nanoscale level in the presence of nanoparticles and a secondary RNA component. Further explorations in vitro and in vivo may contribute to the development of a better cellular delivery platform for Cas9.

Identification, characterization, and verification of miR399 target gene in grape

The microRNA miR399 plays an important role in phosphorus signal transduction pathways in plants. Previously, miR399 was shown to be closely associated with berry ripening in grape (Vitis vinifera). The objective of the present study was to elucidate the evolutionary characteristics of the miR399 gene family in grape and to verify the cleavage effect on the target genes. Grape miR399s were identified by miRNA sequencing and retrieval from the miRBase database. The mature sequences and precursor sequences were subjected to phylogenetic analysis to reconstruct evolutionary trees, as well as secondary structure analysis of the precursor sequence, and prediction of target genes. The cis-acting elements in the miR399 promoter were predicted and the cleavage effect of grape miR399b on its target genes was verified. The grape miR399 family comprised nine precursor sequences and nine mature sequences. The precursor sequences formed a typical and stable stem–loop structure. The minimum folding free energy ranged from −55.70 kcal · mol−1 to −37.40 kcal · mol−1. Multiple sequence alignment revealed that the miR399 family was highly conserved. The grape miR399 family was phylogenetically closely related to peach, apple, and citrus miR399s. Grape miR399s were predicted to target inorganic phosphate transporter 1–3, phospholipase D delta-like, and beta-glucuronosyltransferase. The cleavage effect of grape miR399b on the target genes was verified by means of a dual-luciferase assay and 5′ RLM-RACE. Histochemical GUS staining showed that the promoter activity of miR399b was promoted by GA3 treatment.

Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition.

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5'UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a 'start-stop'. The 'start-stop' sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.

Comparative Mitogenomic Analysis of Heptageniid Mayflies (Insecta: Ephemeroptera): Conserved Intergenic Spacer and tRNA Gene Duplication.

Simple SummaryHeptageniidae is one of the most abundant and widespread families of mayflies, with more than 600 described species and distributed mainly in the Holoarctic, Oriental, and Afrotropical regions. Previous phylogenetic analyses in this family mainly focused on morphological characters and were restricted to one or several genes. Ten complete mitochondrial genomes from this family are reported here. A conserved intergenic spacer and tRNA gene duplication in Heptageniidae mitogenomes may be regarded as ancient features and provide evidence for the phylogenic analysis within Ephemeroptera.Large intergenic spacers and tRNA gene duplications have been reported in several insect groups, although little is known about mitogenomes of mayflies. Here, we determined complete mitogenomes of ten heptageniid species and systemically analyzed their mitogenomic features. Both a conserved intergenic spacer (IGS) and trnM duplication were detected in those mitogenomes. The IGS, which was observed in heptageniids, could be further folded into a stable stem–loop structure. The tRNA gene duplication was found in almost all analyzed mitogenomes, and a unique gene block trnI-trnM-trnQ-trnM-ND2 was also discovered. Our analysis demonstrates that the heptageniid gene arrangement pattern can be explained by the tandem duplication-random loss (TDRL) model. Phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods based on the nucleotide and amino acid sequence data recovered the genus Epeorus as monophyletic with strong support. Our results provide a better understanding of mitogenomic evolution in Heptageniidae, as well as novel molecular markers for species identification of mayflies.

Solution structure and RNA-binding of a minimal ProQ-homolog from Legionella pneumophila (Lpp1663).

Small regulatory RNAs (sRNAs) play an important role for posttranscriptional gene regulation in bacteria. sRNAs recognize their target messenger RNAs (mRNAs) by base-pairing, which is often facilitated by interactions with the bacterial RNA-binding proteins Hfq or ProQ. The FinO/ProQ RNA-binding protein domain was first discovered in the bacterial repressor of conjugation, FinO. Since then, the functional role of FinO/ProQ-like proteins in posttranscriptional gene regulation was extensively studied in particular in the enterobacteria E. coli and Salmonella enterica and a wide range of sRNA-targets was identified for these proteins. In addition, enterobacterial ProQ homologs also recognize and protect the 3′-ends of a number of mRNAs from exonucleolytic degradation. However, the RNA-binding properties of FinO/ProQ proteins with regard to the recognition of different RNA targets are not yet fully understood. Here, we present the solution NMR structure of the so far functionally uncharacterized ProQ homolog Lpp1663 from Legionella pneumophila as a newly confirmed member and a minimal model system of the FinO/ProQ protein family. In addition, we characterize the RNA-binding preferences of Lpp1663 with high resolution NMR spectroscopy and isothermal titration calorimetry (ITC). Our results suggest a binding preference for single-stranded uridine-rich RNAs in the vicinity of stable stem–loop structures. According to chemical shift perturbation experiments, the single-stranded U-rich RNAs interact mainly with a conserved RNA-binding surface on the concave site of Lpp1663.

SncRNA-1 Is a Small Noncoding RNA Produced by Mycobacterium tuberculosis in Infected Cells That Positively Regulates Genes Coupled to Oleic Acid Biosynthesis.

Nearly one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

ABSTRACTPackaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5ʹ untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

The landscape of the non-canonical RNA-binding site of Gemin5 unveils a feedback loop counteracting the negative effect on translation.

Gemin5 is a predominantly cytoplasmic protein that downregulates translation, beyond controlling snRNPs assembly. The C-terminal region harbors a non-canonical RNA-binding site consisting of two domains, RBS1 and RBS2, which differ in RNA-binding capacity and the ability to modulate translation. Here, we show that these domains recognize distinct RNA targets in living cells. Interestingly, the most abundant and exclusive RNA target of the RBS1 domain was Gemin5 mRNA. Biochemical and functional characterization of this target demonstrated that RBS1 polypeptide physically interacts with a predicted thermodynamically stable stem–loop upregulating mRNA translation, thereby counteracting the negative effect of Gemin5 protein on global protein synthesis. In support of this result, destabilization of the stem–loop impairs the stimulatory effect on translation. Moreover, RBS1 stimulates translation of the endogenous Gemin5 mRNA. Hence, although the RBS1 domain downregulates global translation, it positively enhances translation of RNA targets carrying thermodynamically stable secondary structure motifs. This mechanism allows fine-tuning the availability of Gemin5 to play its multiple roles in gene expression control.

Conformational Changes of Single-Stranded Hairpin DNA Covalently Immobilized on Solid Surfaces Investigated by Molecular Dynamics Simulations

Molecular beacons (MBs) are stem-loop hairpin structured nucleic acid probes that have shown greater sensitivity and specificity over linear DNA probes. For high-throughput DNA detection, MBs are often covalently immobilized on a variety of solid surfaces (e.g., glass slides and gold surfaces). The performance of immobilized MBs on solid surfaces has been found to be dependent on the stability and orientation of their stem-loop structures on surfaces. At present, however, there is still a lack of high-resolution experimental characterization approaches for determining the conformational behaviors of immobilized MBs on surfaces. Herein, we investigated the structure and dynamics of immobilized MB probe in NaCl and MgCl2 solutions respectively by performing all-atom molecular dynamics simulations. We found that the single immobilized MB probe maintained a stable stemloop structure in the MgCl2 solution, while it undergone significant structural distortion in the NaCl solution. The results show that Mg2+ ions have a stronger stabilizing effect on immobilized hairpins compared with Na+ ions, which is attributed to their greater shielding ability for the negatively charged phosphate backbone. Moreover, the hydrogen-bonding dynamics in the hairpin's loop and stem parts were analyzed respectively to further understand the effect of the base–base interactions on conformational stability of MBs immobilized on solid surfaces. The findings in this work help to optimize the design of MBs biosensors, thus further improving their performance of nucleic acid detection.

The complete mitochondrial genome of the economic red alga, Gracilaria chilensis

Gracilaria chilensis is an economically important marine alga. In this study, we obtained complete mitogenome of G. chilensis by high-throughput sequencing, which was mapped as a circular molecule of 26,897 bp with 27.56% GC content and was identified 53 genes, including 25 protein-coding genes, 2 rRNA genes, 26 tRNA genes, and 1 group II intron inserted into the trnI gene. In addition, a 162-bp stable stem loop was found in intergenic regions, which was most likely associated with DNA transcription and replication. The Bayesian phylogenetic tree of Gracilariaceae revealed that G. chilensis and G. salicornia and G. changii shared a closer relationship than G. vermiculophylla in the genus Gracilaria.

Genome-wide identification of novel microRNAs from genome sequences using computational approach in the mudskipper (Boleophthalmus pectinirostris)

MicroRNAs (miRNAs), approximately 22 nucleotides (nt) long, are small, non-coding RNA molecules with important regulatory functions in gene expression. They are mostly conserved among the organisms and this conservation makes them a good source for the identification of novel miRNAs by computational genomic homology. The miRNA repertoire of a major aquaculture species, Boleophthalmus pectinirostris, has been unknown until recently. Currently, the B. pectinirostris whole-genome sequences have been completed, making it more convenient for us to focus on computational prediction for novel miRNA homologs. Following a range of strict filtering criteria, a total of 62 potential miRNAs were identified for the first time; they belong to 39 different miRNA families. All these miRNAs were observed in the stem portion of the stable stem–loop structures. The minimum free energy (MFE) of the predicted miRNAs ranged from −21.6 to −62.7 kcal/mol with an average of −39.2 kcal/mol. The A + U ranged from 32.5 to 69.1% with an average value of 52.2%. The phylogenetic analysis of predicted miRNAs revealed that miR-23a-3p, miR-184-3p, miR-214-5p, and miR-338-3p from B. pectinirostris are evolutionary highly conserved showing more similarity with other fish species. To verify the predicted miRNAs, selected miRNAs representing 16 of the 39 families were confirmed by stem–loop RT-PCR, indicating that the computational approach that we used to identify the miRNAs is a highly efficient and affordable alternative method. Taken together, these findings provide a reference point for further research on miRNAs identification in fish species, meanwhile, our study also will be useful for further insight into biological functions of miRNAs and improved understanding of genome in B. pectinirostris.

The complete mitochondrial genome of Plodia interpunctella (Lepidoptera: Pyralidae) and comparison with other Pyraloidea insects.

The mitochondrial (mt) genome can provide important information for the understanding of phylogenetic relationships. The complete mt genome of Plodia interpunctella (Lepidoptera: Pyralidae) has been sequenced. The circular genome is 15 287 bp in size, encoding 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The AT skew of this mt genome is slightly negative, and the nucleotide composition is biased toward A+T nucleotides (80.15%). All PCGs start with the typical ATN (ATA, ATC, ATG, and ATT) codons, except for the cox1 gene which may start with the CGA codon. Four of the 13 PCGs harbor the incomplete termination codon T or TA. All the tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNA, except for trnS1 (AGN) in which the DHU arm fails to form a stable stem-loop structure. The overlapping sequences are 35 bp in total and are found in seven different locations. A total of 240 bp of intergenic spacers are scattered in 16 regions. The control region of the mt genome is 327 bp in length and consisted of several features common to the sequenced lepidopteran insects. Phylogenetic analysis based on 13 PCGs using the Maximum Likelihood method shows that the placement of P. interpunctella was within the Pyralidae.

HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site.

The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing regulatory (SR) proteins, in particular SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins.

Intron retention in the 5'UTR of the novel ZIF2 transporter enhances translation to promote zinc tolerance in arabidopsis.

Root vacuolar sequestration is one of the best-conserved plant strategies to cope with heavy metal toxicity. Here we report that zinc (Zn) tolerance in Arabidopsis requires the action of a novel Major Facilitator Superfamily (MFS) transporter. We show that ZIF2 (Zinc-Induced Facilitator 2) localises primarily at the tonoplast of root cortical cells and is a functional transporter able to mediate Zn efflux when heterologously expressed in yeast. By affecting plant tissue partitioning of the metal ion, loss of ZIF2 function exacerbates plant sensitivity to excess Zn, while its overexpression enhances Zn tolerance. The ZIF2 gene is Zn-induced and an intron retention event in its 5′UTR generates two splice variants (ZIF2.1 and ZIF2.2) encoding the same protein. Importantly, high Zn favours production of the longer ZIF2.2 transcript, which compared to ZIF2.1 confers greater Zn tolerance to transgenic plants by promoting higher root Zn immobilization. We show that the retained intron in the ZIF2 5′UTR enhances translation in a Zn-responsive manner, markedly promoting ZIF2 protein expression under excess Zn. Moreover, Zn regulation of translation driven by the ZIF2.2 5′UTR depends largely on a predicted stable stem loop immediately upstream of the start codon that is lost in the ZIF2.1 5′UTR. Collectively, our findings indicate that alternative splicing controls the levels of a Zn-responsive mRNA variant of the ZIF2 transporter to enhance plant tolerance to the metal ion.

UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells

UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.

Deciphering Ribosomal Frameshifting Dynamics

Ribosomes programmed by specific messenger RNA (mRNA) sequence elements can switch translation reading frames and synthesize different polypeptides from a single template. The Escherichia coli dnaX mRNA encodes two DNA polymerase III subunits, τ and γ, synthesized from 0-frame and probabilistic −1-slip across the slippery sequence: AAAAAAG. When further enhanced by structural barriers situated around the slippery sequence-an internal Shine-Dalgarno sequence and a stable hairpin stem loop, an 80% (= γ/(γ+τ)) frameshift efficiency is attained. Here, we attempt to determine the frameshift timing within one translation cycle by following a single ribosome translating a frameshift-promoting mRNA held on optical tweezers. In parallel, by mass spectrometry (MS), we survey the synthesized polypeptides to resolve where on the mRNA the ribosome has slipped.From the mass spectra of polypeptides terminated at the −1-frame stop codon, we learned that the ribosome −1-slips from more than one codon position around the slippery sequence. Some −1-frameshifted polypeptides were found to bear an extra amino acid, or to lack one, indicating that slipping sizes of −4 and +2-nt also occurred. Similarly, distinctive large-scale fluctuating translocation dynamics were seen in our real-time single-ribosome translation trajectories. This reveals that a translocating ribosome can explore a broad range of frameshift pathways. Frequently adopted frameshift pathways, i.e. the more abundant frameshifted species resolved by MS, exhibit a preference for minimizing codon:anticodon base-pair mismatches on the ribosome after a slip. Mismatch-containing ribosomes can be prematurely terminated by release factors, resulting in release of incomplete peptides. Indeed, we observed higher yields of incomplete peptides that are terminated at frameshift sites where significant mismatches were encountered. These species coincide with the prematurely stalled ribosomes recorded in the translation trajectories. Collectively what emerges from our results is a versatile ribosomal frameshifting scheme during mRNA translocation, facilitating branching of frameshift pathways.

Two-Dimensional Electrophoresis-Based Selection of Aptamers Against an Unidentified Protein in a Tissue Sample

A fully automated two-dimensional electrophoresis (2DE) system was employed for DNA aptamer selection against an unidentified protein in a mouse liver tissue extract as a model target. A 2DE-based systematic evolution of ligands by exponential enrichment (2DE-SELEX) was demonstrated for aptamer selection against a single protein spot that was separated on a nitrocellulose membrane. After four iterative 2DE-SELEX cycles, the oligonucleotide pool was sequenced and aptamer sequences were identified. A blotting assay showed that an identified aptamer with a stable stem–loop structure had specific binding activity against the target protein. The 2DE-SELEX was shown to be promising for the development of aptamers against unidentified proteins in complex samples for proteomic analysis and biomarker discovery. Supplemental materials are available for this article. Go to the publisher's online edition of Analytical Letters to view the supplemental file.

Description of Nearly Completed Mitochondrial Genome Sequences of the Garden Chafer Polyphylla laticollis manchurica, Endangered in Korea (Insecta: Coleoptera)

In this study, we present the nearly complete mitogenome sequences of the garden chafer, Polyphylla laticollis manchurica, which is listed as an endangered species in Korea. The P. l. manchurica mitogenome, which includes unfinished whole A+T-rich region and a partial srRNA was 14,473-bp long, possessing typical sets of genes (13 PCGs, 22 tRNA genes, and 2 rRNA genes). Gene arrangement of the P. l. manchurica mitogenome was identical to the common one found in the majority of insects. The 5 bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand was also found in the P. l. manchurica mitogenome between <TEX>$tRNA^{Ser}$</TEX>(UCN) and ND1. The start codon for COI gene and ATPase8 was designated as a typical TTG. All tRNAs of the P. l. manchurica showed a stable canonical clover-leaf structure of other mt tRNAs, except for <TEX>$tRNA^{Ser}$</TEX>(AGN), DHU arm of which could not form stable stemloop structure. As has been previously determined, the high A/T content was unanimously observed in P. l. manchurica in terms of A/T bias in the third codon position (73.5%) compared with the first (66.4%) and second codon position (66.2%). The PCGs encoded in major-strands are slightly T-skewed, whereas those of the minor-strand are A-skewed, indicating strand asymmetry in nucleotide composition in the Coleoptera including P. l. manchurica.

5′-UTR mediated translational control of splicing assembly factor RNP-4F expression during development of the Drosophila central nervous system

Drosophila RNP-4F is a highly conserved protein from yeast to human and functions as a spliceosome assembly factor during pre-mRNA splicing. Two major developmentally regulated rnp-4f mRNA isoforms have been described during fly development, designated “long” and “short,” differing by a 177-nt tract in the 5′-UTR. This region potentially folds into a single long stable stem–loop by pairing of intron 0 and part of exon 2. Since the coding potential for the two isoforms is identical, the interesting question arises as to the functional significance of this evolutionarily-conserved 5′-UTR feature. Here we describe the effects of wild-type and mutated stem–loop on modulation of rnp-4f gene expression in embryos using a GFP reporter assay. In this work, a new GFP expression vector designated pUAS-Neostinger was constructed. The UAS-GAL4 system was utilized to trigger GFP expression using tissue-specific promoter driver fly lines. Fluorescence microscopy visualization, Western blotting and real-time qRT-PÇR were used to study and quantify GFP reporter protein and mRNA levels. A significant increase in GFP reporter protein expression due to presence of the wild-type stem–loop sequence/structure was unexpectedly observed with no concomitant increase in GFP reporter mRNA levels, showing that the 177-nt region enhancement acts posttranscriptionally. The effects of potential cis-acting elements within the stem–loop were evaluated using the reporter assay in two mutant constructs. Results of GFP reporter over-expression show that RNP-4F translational regulation is highly sensitive in the developing fly central nervous system. The potential molecular mechanism behind the observed translational enhancement is discussed.

Molecular characterizations of two mitoviruses co-infecting a hyovirulent isolate of the plant pathogenic fungus Sclerotinia sclerotiorum

The complete nucleotide sequences of two double-stranded RNA (dsRNA) segments, isolated from the same hypovirulent strain (KL-1) of Sclerotinia sclerotiorum, were determined. Sequence analysis showed that dsRNAs 1 to be 2513 nts long and is A–U rich (61.7%). Excluding the poly(A) tail, dsRNAs2 is 2421 nts long and its AU content is 53.1%. The 5′ and 3′-terminal sequences of the positive-strand of each dsRNA could be folded into predicted stable stem–loop structures. Mitochondrial codon usage revealed that each dsRNA has a single large open reading frame coding for a protein containing RNA-dependent RNA polymerase conserved motifs. Furthermore, dsRNAs 1 and 2 share sequence similarities with other mitoviruses. These results suggest that dsRNAs 1 and 2 represent two distinct new mitoviruses, designated Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL-1) and SsMV2/KL-1, respectively. The hypovirulence traits of strain KL-1 and the two mitoviruses could be co-transmitted to a virus-free virulent strain via hyphal anastomosis.