To understand an RNA sequence's mechanism of action, the structure must be known. Furthermore, target RNA structure is an important consideration in the design of small interfering RNAs and antisense DNA oligonucleotides. RNA secondary structure prediction, using thermodynamics, can be used to develop hypotheses about the structure of an RNA sequence. RNAstructure is a software package for RNA secondary structure prediction and analysis. It uses thermodynamics and utilizes the most recent set of nearest neighbor parameters from the Turner group. It includes methods for secondary structure prediction (using several algorithms), prediction of base pair probabilities, bimolecular structure prediction, and prediction of a structure common to two sequences. This contribution describes new extensions to the package, including a library of C++ classes for incorporation into other programs, a user-friendly graphical user interface written in JAVA, and new Unix-style text interfaces. The original graphical user interface for Microsoft Windows is still maintained. The extensions to RNAstructure serve to make RNA secondary structure prediction user-friendly. The package is available for download from the Mathews lab homepage at http://rna.urmc.rochester.edu/RNAstructure.html
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/pdf/rnastructure-software-for-rna-secondary-structure-prediction-3e42o4sg6f.pdf

RNAstructure: software for RNA secondary structure prediction and analysis

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS and other serious health threats. Viral replication is regulated at many levels, including the use of conserved genomic RNA structures. Most potential regulatory elements in viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests that RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions, including splice site acceptors and hypervariable regions. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by previously unrecognized regulatory motifs and that extensive RNA structure constitutes an important component of the genetic code.

/pdf/architecture-and-secondary-structure-of-an-entire-hiv-1-rna-42sm288fkr.pdf

Architecture and secondary structure of an entire HIV-1 RNA genome

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7-kilobase lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high messenger RNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a range of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25-nucleotide 'TINCR box' motif that is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyse TINCR binding capacity to approximately 9,400 human recombinant proteins revealed direct binding of TINCR RNA to the staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay, however, did not have differentiation effects. Instead, the TINCR-STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through lncRNA binding to differentiation mRNAs to ensure their expression.

Control of somatic tissue differentiation by the long non-coding RNA TINCR

RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational and experimental efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo.

Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo

RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

/pdf/in-vivo-genome-wide-profiling-of-rna-secondary-structure-1mojowj6bf.pdf

In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features

Almost all RNAs can fold to form extensive base-paired secondary structures. Many of these structures then modulate numerous fundamental elements of gene expression. Deducing these structure–function relationships requires that it be possible to predict RNA secondary structures accurately. However, RNA secondary structure prediction for large RNAs, such that a single predicted structure for a single sequence reliably represents the correct structure, has remained an unsolved problem. Here, we demonstrate that quantitative, nucleotide-resolution information from a SHAPE experiment can be interpreted as a pseudo-free energy change term and used to determine RNA secondary structure with high accuracy. Free energy minimization, by using SHAPE pseudo-free energies, in conjunction with nearest neighbor parameters, predicts the secondary structure of deproteinized Escherichia coli 16S rRNA (>1,300 nt) and a set of smaller RNAs (75–155 nt) with accuracies of up to 96–100%, which are comparable to the best accuracies achievable by comparative sequence analysis.

https://www.pnas.org/content/pnas/106/1/97.full.pdf

Accurate SHAPE-directed RNA structure determination

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 29-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 29-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 29-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.

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Katherine E. Deigan

Papers

Accurate SHAPE-directed RNA structure determination

Influence of nucleotide identity on ribose 2′-hydroxyl reactivity in RNA

Accurate SHAPE‐directed RNA structure prediction