Evidence for the existence of the loop E motif of Potato spindle tuber viroid in vivo

RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid. We present our findings and discuss their biological implications.     

Using binding profiles to predict binding sites of target RNAs

Prediction of RNA-RNA interaction is a key to elucidating possible functions of small non-coding RNAs, and a number of computational methods have been proposed to analyze interacting RNA secondary structures. In this article, we focus on predicting binding sites of target RNAs that are expected to interact with regulatory antisense RNAs in a general form of interaction. For this purpose, we propose bistaRNA, a novel method for predicting multiple binding sites of target RNAs. bistaRNA employs binding profiles that represent scores for hybridized structures, leading to reducing the computational cost for interaction prediction. bistaRNA considers an ensemble of equilibrium interacting structures and seeks to maximize expected accuracy using dynamic programming. Experimental results on real interaction data validate good accuracy and fast computation time of bistaRNA as compared with several competitive methods. Moreover, we aim to find new targets given specific antisense RNAs, which provides interesting insights into antisense RNA regulation. bistaRNA is implemented in C++. The program and Supplementary Material are available at http://rna.naist.jp/program/bistarna/.     

Epitranscriptomic technologies and analyses

RNA can interact with RNA-binding proteins(RBPs), mRNA, or other non-coding RNAs(ncRNAs) to form complex regulatory networks. High-throughput CLIP-seq, degradome-seq, and RNA-RNA interactome sequencing methods represent powerful approaches to identify biologically relevant ncRNA-target and protein-ncRNA interactions. However, assigning ncRNAs to their regulatory target genes or interacting RNA-binding proteins(RBPs) remains technically challenging. Chemical modifications to mRNA also play important roles in regulating gene expression. Investigation of the functional roles of these modifications relies highly on the detection methods used. RNA structure is also critical at nearly every step of the RNA life cycle. In this review, we summarize recent advances and limitations in CLIP technologies and discuss the computational challenges of and bioinformatics tools used for decoding the functions and regulatory networks of ncRNAs. We also summarize methods used to detect RNA modifications and to probe RNA structure.     

Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.     

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.     

Thermodynamics of RNA-RNA binding

BACKGROUND: Reliable prediction of RNA-RNA binding energies is crucial, e.g. for the understanding on RNAi, microRNA-mRNA binding and antisense interactions. The thermodynamics of such RNA-RNA interactions can be understood as the sum of two energy contributions: (1) the energy necessary to 'open' the binding site and (2) the energy gained from hybridization. METHODS: We present an extension of the standard partition function approach to RNA secondary structures that computes the probabilities Pu[i, j] that a sequence interval [i, j] is unpaired. RESULTS: Comparison with experimental data shows that Pu[i, j] can be applied as a significant determinant of local target site accessibility for RNA interference (RNAi). Furthermore, these quantities can be used to rigorously determine binding free energies of short oligomers to large mRNA targets. The resource consumption is comparable with a single partition function computation for the large target molecule. We can show that RNAi efficiency correlates well with the binding energies of siRNAs to their respective mRNA target. AVAILABILITY: RNAup will be distributed as part of the Vienna RNA Package, www.tbi.univie.ac.at/~ivo/RNA/     

Deciphering protein-protein interactions. Part II. Computational methods to predict protein and domain interaction partners

Recent advances in high-throughput experimental methods for the identification of protein interactions have resulted in a large amount of diverse data that are somewhat incomplete and contradictory. As valuable as they are, such experimental approaches studying protein interactomes have certain limitations that can be complemented by the computational methods for predicting protein interactions. In this review we describe different approaches to predict protein interaction partners as well as highlight recent achievements in the prediction of specific domains mediating protein-protein interactions. We discuss the applicability of computational methods to different types of prediction problems and point out limitations common to all of them.     

A balance between circular and linear forms of the dengue virus genome is crucial for viral replication

The plasticity of viral plus strand RNA genomes is fundamental for the multiple functions of these molecules. Local and long-range RNA-RNA interactions provide the scaffold for interacting proteins of the translation, replication, and encapsidation machinery. Using dengue virus as a model, we investigated the relevance of the interplay between two alternative conformations of the viral genome during replication. Flaviviruses require long-range RNA-RNA interactions and genome cyclization for RNA synthesis. Here, we define a sequence present in the viral 3'UTR that overlaps two mutually exclusive structures. This sequence can form an extended duplex by long-range 5'-3' interactions in the circular conformation of the RNA or fold locally into a small hairpin (sHP) in the linear form of the genome. A mutational analysis of the sHP structure revealed an absolute requirement of this element for viral viability, suggesting the need of a linear conformation of the genome. Viral RNA replication showed high vulnerability to changes that alter the balance between circular and linear forms of the RNA. Mutations that shift the equilibrium toward the circular or the linear conformation of the genome spontaneously revert to sequences with different mutations that tend to restore the relative stability of the two competing structures. We propose a model in which the viral genome exists in at least two alternative conformations and the balance between these two states is critical for infectivity.     

Partner-specific prediction of RNA-binding residues in proteins: A critical assessment

RNA-protein interactions play essential roles in regulating gene expression. While some RNA-protein interactions are “specific”, that is, the RNA-binding proteins preferentially bind to particular RNA sequence or structural motifs, others are “non-RNA specific.” Deciphering the protein-RNA recognition code is essential for comprehending the functional implications of these interactions and for developing new therapies for many diseases. Because of the high cost of experimental determination of protein-RNA interfaces, there is a need for computational methods to identify RNA-binding residues in proteins. While most of the existing computational methods for predicting RNA-binding residues in RNA-binding proteins are oblivious to the characteristics of the partner RNA, there is growing interest in methods for partner-specific prediction of RNA binding sites in proteins. In this work, we assess the performance of two recently published partner-specific protein-RNA interface prediction tools, PS-PRIP, and PRIdictor, along with our own new tools. Specifically, we introduce a novel metric, RNA-specificity metric (RSM), for quantifying the RNA-specificity of the RNA binding residues predicted by such tools. Our results show that the RNA-binding residues predicted by previously published methods are oblivious to the characteristics of the putative RNA binding partner. Moreover, when evaluated using partner-agnostic metrics, RNA partner-specific methods are outperformed by the state-of-the-art partner-agnostic methods. We conjecture that either (a) the protein-RNA complexes in PDB are not representative of the protein-RNA interactions in nature, or (b) the current methods for partner-specific prediction of RNA-binding residues in proteins fail to account for the differences in RNA partner-specific versus partner-agnostic protein-RNA interactions, or both.     

Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosine-mediated photocrosslinking.

Expression of the structural proteins of human immunodeficiency virus type 1 (HIV-1) requires the direct interaction of multiple copies of the viral protein Rev with its target RNA, the Rev response element (RRE). RRE is a complex 351-nt RNA that is highly structured and located within the viral env gene. During initial Rev-RRE recognition, Rev binds with high affinity to a bubble structure located within the RRE RNA stem-loop II. We have used a site-specific photocrosslinking method based on 6-thioguanosine (6-thioG) photochemistry to probe the conformation of the high-affinity binding site of RRE RNA and its interactions with Rev protein under physiological conditions. A minimal duplex RNA containing the bubble region of RRE and 12 flanking base pairs was synthesized chemically. Two different RRE constructs with a single photoactive nucleoside (6-thio-dG or 6-thioG) at position 47 or 48 were synthesized. Upon UV irradiation, 6-thioG at both positions formed interstrand covalent crosslinks in RRE RNA. Mapping of crosslink sites by RNA sequencing revealed that 6-thioG at position 47 or 48 crosslinked to A73. In the presence of Rev, both RNA-RNA and RNA-protein crosslinks were observed, however, the RNA-RNA crosslink site was unchanged. Our results provide direct evidence that, during RNA-protein recognition, Rev is in close proximity to O6 of G47 and G48 in the major groove of RRE RNA. Our results also show that the bubble region of RRE RNA has a biologically relevant structure where G47 and G48 are in close proximity to A73 and this RNA structure is not changed significantly upon Rev binding. We propose that Rev protein recognizes and binds to specific structural elements of RRE RNA containing non-Watson-Crick base pairs and such structures could be a determinant for recognition by other RNA-binding proteins. Our site-specific crosslinking methods provide a general approach to capture dynamic states of biologically relevant RNA structures that are otherwise missed by NMR and X-ray crystallographic studies.     

Protein–DNA/RNA interactions: Machine intelligence tools and approaches in the era of artificial intelligence and big data

With the development of artificial intelligence (AI) technologies and the availability of large amounts of biological data, computational methods for proteomics have undergone a developmental process from traditional machine learning to deep learning. This review focuses on computational approaches and tools for the prediction of protein – DNA/RNA interactions using machine intelligence techniques. We provide an overview of the development progress of computational methods and summarize the advantages and shortcomings of these methods. We further compiled applications in tasks related to the protein – DNA/RNA interactions, and pointed out possible future application trends. Moreover, biological sequence-digitizing representation strategies used in different types of computational methods are also summarized and discussed.     

RNA pseudoknot prediction in energy-based models.

RNA molecules are sequences of nucleotides that serve as more than mere intermediaries between DNA and proteins, e.g., as catalytic molecules. Computational prediction of RNA secondary structure is among the few structure prediction problems that can be solved satisfactorily in polynomial time. Most work has been done to predict structures that do not contain pseudoknots. Allowing pseudoknots introduces modeling and computational problems. In this paper we consider the problem of predicting RNA secondary structures with pseudoknots based on free energy minimization. We first give a brief comparison of energy-based methods for predicting RNA secondary structures with pseudoknots. We then prove that the general problem of predicting RNA secondary structures containing pseudoknots is NP complete for a large class of reasonable models of pseudoknots.     

Sequence-specific binding of single-stranded RNA: is there a code for recognition?

A code predicting the RNA sequence that will be bound by a certain protein based on its amino acid sequence or its structure would provide a useful tool for the design of RNA binders with desired sequence-specificity. Such de novo designed RNA binders could be of extraordinary use in both medical and basic research applications. Furthermore, a code could help to predict the cellular functions of RNA-binding proteins that have not yet been extensively studied. A comparative analysis of Pumilio homology domains, zinc-containing RNA binders, hnRNP K homology domains and RNA recognition motifs is performed in this review. Based on this, a set of binding rules is proposed that hints towards a code for RNA recognition by these domains. Furthermore, we discuss the intermolecular interactions that are important for RNA binding and summarize their importance in providing affinity and specificity.