Identification and Characterization of Evolutionary Conserved Muskmelon Non-coding miRNAs and Their Target Proteins.

MicroRNAs (miRNAs) are short, endogenously encoded small RNAs, 18-26 nucleotides in length, which can posttranscriptionally regulate gene expression through translation inhibition or endonucleolytic cleavage. The muskmelon is one of the most widely cultivated fruits in the Cucurbitaceae family. Despite its significance, only 120 miRNAs from different families have been reported in muskmelon. In this study, we aimed to expand this knowledge base by predicting 40 new miRNAs in muskmelon using a spectrum of genomic-based tools. Precursor and mature sequences were obtained from microRNA registry database as reference and analyzed via the basic local alignment search tool (Blastn) for ESTs identification. After removing the non-coding sequences, the remaining candidate sequences were analyzed using MFOLD to generate secondary structures for the newly predicted miRNAs. Additionally, the predicted muskmelon miRNAs were validated using a set of five randomly chosen primers and RT-PCR. Through gene ontology (GO) analysis, we identified 89 targets associated with newly predicted muskmelon miRNAs. Transcription factor-coding genes play a crucial role in plant growth and development. Additionally, the miR4249 has been found to have the same targets in muskmelon that have been linked to cell signaling and transcription factors. The identified targets are integral for diverse biological processes including plant growth, development, metabolism, aging, disease resistance, and resistance to environmental stresses, such as salt, cold, and oxidative stress. As a result, the outcomes of this study demonstrate that this mechanism not only contributes to the production of a higher quality crop but also enhances overall production.

The Secondary Structure of Potato Spindle Tuber Viroid Determines Its Infectivity in Nicotiana benthamiana.

The function of RNAs is determined by their structure. However, studying the relationship between RNA structure and function often requires altering RNA sequences to modify the structures, which leads to the neglect of the importance of RNA sequences themselves. In our research, we utilized potato spindle tuber viroid (PSTVd), a circular-form non-coding infectious RNA, as a model with which to investigate the role of a specific rod-like structure in RNA function. By generating linear RNA transcripts with different start sites, we established 12 PSTVd forms with different secondary structures while maintaining the same sequence. The RNA secondary structures were predicted using the mfold tool and validated through native PAGE gel electrophoresis after in vitro RNA folding. Analysis using plant infection assays revealed that the formation of a correct rod-like structure is crucial for the successful infection of PSTVd. Interestingly, the inability of PSTVd forms with non-rod-like structures to infect plants could be partially compensated by increasing the amount of linear viroid RNA transcripts, suggesting the existence of additional RNA secondary structures, such as the correct rod-like structure, alongside the dominant structure in the RNA inoculum of these forms. Our study demonstrates the critical role of RNA secondary structures in determining the function of infectious RNAs.

Inverse-Folding Design of Yeast Telomerase RNA Increases Activity In Vitro.

Saccharomyces cerevisiae telomerase RNA, TLC1, is an 1157 nt non-coding RNA that functions as both a template for DNA synthesis and a flexible scaffold for telomerase RNP holoenzyme protein subunits. The tractable budding yeast system has provided landmark discoveries about telomere biology in vivo, but yeast telomerase research has been hampered by the fact that the large TLC1 RNA subunit does not support robust telomerase activity in vitro. In contrast, 155-500 nt miniaturized TLC1 alleles comprising the catalytic core domain and lacking the RNA's long arms do reconstitute robust activity. We hypothesized that full-length TLC1 is prone to misfolding in vitro. To create a full-length yeast telomerase RNA, predicted to fold into its biologically relevant structure, we took an inverse RNA-folding approach, changing 59 nucleotides predicted to increase the energetic favorability of folding into the modeled native structure based on the p-num feature of Mfold software. The sequence changes lowered the predicted ∆G of this "determined-arm" allele, DA-TLC1, by 61 kcal/mol (-19%) compared to wild-type. We tested DA-TLC1 for reconstituted activity and found it to be ~5-fold more robust than wild-type TLC1, suggesting that the inverse-folding design indeed improved folding in vitro into a catalytically active conformation. We also tested if DA-TLC1 functions in vivo, discovering that it complements a tlc1∆ strain, allowing cells to avoid senescence and maintain telomeres of nearly wild-type length. However, all inverse-designed RNAs that we tested had reduced abundance in vivo. In particular, inverse-designing nearly all of the Ku arm caused a profound reduction in telomerase RNA abundance in the cell and very short telomeres. Overall, these results show that the inverse design of S. cerevisiae telomerase RNA increases activity in vitro, while reducing abundance in vivo. This study provides a biochemically and biologically tested approach to inverse-design RNAs using Mfold that could be useful for controlling RNA structure in basic research and biomedicine.

Significant Differences in RNA Structure Destabilization by HIV-1 GagDp6 and NCp7 Proteins.

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play distinct roles in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structures, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a complementary RNA hairpin. In contrast, during viral assembly, NC, as a domain of the group-specific antigen (Gag) polyprotein, binds the genomic RNA and facilitates packaging into new virions. It is not clear how the same protein, alone or as part of Gag, performs such different RNA binding functions in the viral life cycle. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium stability and unfolding barrier for TAR RNA. Comparing measured results with a model of discrete protein binding allows us to localize affected binding sites, in addition to quantifying hairpin stability. We find that, while both NCp7 and GagDp6 destabilize the TAR hairpin, GagDp6 binding is localized to two sites in the stem, while NCp7 targets sites near the top loop. Unlike GagDp6, NCp7 destabilizes this loop, shifting the location of the reaction barrier toward the folded state and increasing the natural rate of hairpin opening by ~104. Thus, our results explain why Gag cleavage and NC release is an essential prerequisite for reverse transcription within the virion.

Specific Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein Deduced from Hairpin Unfolding.

RNA and DNA hairpin formation and disruption play key regulatory roles in a variety of cellular processes. The 59-nucleotide transactivation response (TAR) RNA hairpin facilitates the production of full-length transcripts of the HIV-1 genome. Yet the stability of this long, irregular hairpin becomes a liability during reverse transcription as 24 base pairs must be disrupted for strand transfer. Retroviral nucleocapsid (NC) proteins serve as nucleic acid chaperones that have been shown to both destabilize the TAR hairpin and facilitate strand annealing with its complementary DNA sequence. Yet it has remained difficult to elucidate the way NC targets and dramatically destabilizes this hairpin while only weakly affecting the annealed product. In this work, we used optical tweezers to measure the stability of TAR and found that adding NC destabilized the hairpin and simultaneously caused a distinct change in both the height and location of the energy barrier. This data was matched to an energy landscape predicted from a simple theory of definite base pair destabilization. Comparisons revealed the specific binding sites found by NC along the irregular TAR hairpin. Furthermore, specific binding explained both the unusual shift in the transition state and the much weaker effect on the annealed product. These experiments illustrate a general method of energy landscape transformation that exposes important physical insights.

Constructing Free Energy Landscapes of Nucleic Acid Hairpin Unfolding.

Single nucleic acid molecules form hairpins that may stabilize secondary and tertiary structures as well as perform enzymatic and other chemical functions. Considerable progress has been made in the effort to understand the contributions of various factors to the stability of a given hairpin sequence. For a given sequence, it is possible to compute both the most likely structural arrangements and their associated free energies over a range of experimental conditions. However, there are many observed hairpin irregularities for which the energies and function are not well understood. Here we examine the irregular RNA Transactivation Response (TAR) hairpin from the HIV-1 genome. Using single molecule optical tweezers, the hairpin is force unfolded, revealing the overall unfolding free energy and the character of the transition state. These measurements allow the construction of a simple energy landscape from unfolding measurements, which can be directly compared to a theoretical landscape. This method is easily adapted to other structures, including the effects of noncanonical bases and even ligand binding.

Influence of gag and RRE Sequences on HIV-1 RNA Packaging Signal Structure and Function.

The packaging signal (Ψ) and Rev-responsive element (RRE) enable unspliced HIV-1 RNAs' export from the nucleus and packaging into virions. For some retroviruses, engrafting Ψ onto a heterologous RNA is sufficient to direct encapsidation. In contrast, HIV-1 RNA packaging requires 5' leader Ψ elements plus poorly defined additional features. We previously defined minimal 5' leader sequences competitive with intact Ψ for HIV-1 packaging, and here examined the potential roles of additional downstream elements. The findings confirmed that together, HIV-1 5' leader Ψ sequences plus a nuclear export element are sufficient to specify packaging. However, RNAs trafficked using a heterologous export element did not compete well with RNAs using HIV-1's RRE. Furthermore, some RNA additions to well-packaged minimal vectors rendered them packaging-defective. These defects were rescued by extending gag sequences in their native context. To understand these packaging defects' causes, in vitro dimerization properties of RNAs containing minimal packaging elements were compared to RNAs with sequence extensions that were or were not compatible with packaging. In vitro dimerization was found to correlate with packaging phenotypes, suggesting that HIV-1 evolved to prevent 5' leader residues' base pairing with downstream residues and misfolding of the packaging signal. Our findings explain why gag sequences have been implicated in packaging and show that RRE's packaging contributions appear more specific than nuclear export alone. Paired with recent work showing that sequences upstream of Ψ can dictate RNA folds, the current work explains how genetic context of minimal packaging elements contributes to HIV-1 RNA fate determination.

Identification of conserved miRNA molecules in einkorn wheat (Triticum monococcum subsp. monococcum) by using small RNA sequencing analysis.

Triticum monococcum subsp. monococcum as a first cultivated diploid wheat species possesses desirable agronomic and quality characteristics. Drought and salinity are the most dramatic environmental stress factors that have serious impact on yield and quality of crops; however, plants can use alternative defense mechanisms against these stresses. The posttranscriptional alteration of gene expression by microRNAs (miRNAs) is one of the most conserved mechanisms. In plant species including wheat genomes, miRNAs have been implicated in the management of salt and drought stress; however, studies on einkorn wheat (Triticum monococcum subsp. monococcum) are not yet available. In this study, we aimed to identify conserved miRNAs in einkorn wheat using next generation sequencing technology and bioinformatics analysis. In order to include a larger set of miRNAs, small RNA molecules from pooled plant samples grown under normal, drought, and salinity conditions were used for the library preparation and sequence analysis. After bioinformatics analysis, we identified 167 putative mature miRNA sequences belonging to 140 distinct miRNA families. We also presented a comparative analysis to propose that miRNAs and their target genes were involved in salt and drought stress control in addition to a comprehensive analysis of the scanned target genes in the T. aestivum genome.

Rational design of a structure-switching DNA aptamer for potassium ions.

Structure-switching molecules provide a unique means for analyte detection, generating a response to analyte concentration through a binding-specific conformational change between non-binding and binding-competent states. While most ligand-binding molecules are not structure switching by default, many can be engineered to be so through the introduction of an alternative non-binding (and thus non-signalling) conformation. This population-shift mechanism is particularly effective with oligonucleotides and has led to the creation of structure-switching aptamers for many target ligands. Here, we report the rational design of structure-switching DNA aptamers, based on the thrombin binding aptamer (TBA), that bind potassium with affinities that bridge the gap between previously reported weak-binding and strong-binding aptamers. We also demonstrate a correlation between the free energy of the experimentally determined binding affinity for potassium and the computationally estimated free energy of the alternative (non-binding) structure.

Systematic analysis of the role of target site accessibility in the activity of DNA enzymes.

We employed an approach using oligonucleotide scanning arrays and computational analysis to conduct a systematic analysis of the interaction between catalytic nucleic acids (DNA enzymes or DNAzymes) and long RNA targets. A radio-labelled transcript representing mRNA of Xenopus cyclin B5 was hybridised to an array of oligonucleotides scanning the first 120 nucleotides of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with the target. The hybridisation revealed oligonucleotides showing varying levels of signal intensities along the length of the array, reflecting on the variable accessibility of the corresponding complementary regions in the target RNA. Deoxyribozymes targeting a number of these regions were selected and tested for their ability to cleave the target RNA. The mRNA cleavage observed indicates that indeed target accessibility was an important component in the activity of deoxyribozymes and that it was important that at least one of the two binding arms was complementary to an accessible site. Computational analysis suggested that intra-molecular folding of deoxyribozymes into stable structures may also negatively contribute to their activity. 10-23 type deoxyribozymes generally appeared more active than 8-17 type and it was possible to predict deoxyribozymes with high cleavage efficiency using scanning array hybridization and computational analysis as guides. The data presented here therefore have implications on designing effective DNA enzymes.