Using SHAPE-MaP To Model RNA Secondary Structure and Identify 3'UTR Variation in Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.

Structural divergence creates new functional features in alphavirus genomes.

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins.

A Functional riboSNitch in the 3' Untranslated Region of FKBP5 Alters MicroRNA-320a Binding Efficiency and Mediates Vulnerability to Chronic Post-Traumatic Pain.

Previous studies have shown that common variants of the gene coding for FK506-binding protein 51 (FKBP5), a critical regulator of glucocorticoid sensitivity, affect vulnerability to stress-related disorders. In a previous report, FKBP5 rs1360780 was identified as a functional variant because of its effect on gene methylation. Here we report evidence for a novel functional FKBP5 allele, rs3800373. This study assessed the association between rs3800373 and post-traumatic chronic pain in 1607 women and men from two ethnically diverse human cohorts. The molecular mechanism through which rs3800373 affects adverse outcomes was established via in silico, in vivo, and in vitro analyses. The rs3800373 minor allele predicted worse adverse outcomes after trauma exposure, such that individuals with the minor (risk) allele developed more severe post-traumatic chronic musculoskeletal pain. Among these individuals, peritraumatic circulating FKBP5 expression levels increased as cortisol and glucocorticoid receptor (NR3C1) mRNA levels increased, consistent with increased glucocorticoid resistance. Bioinformatic, in vitro, and mutational analyses indicate that the rs3800373 minor allele reduces the binding of a stress- and pain-associated microRNA, miR-320a, to FKBP5 via altering the FKBP5 mRNA 3'UTR secondary structure (i.e., is a riboSNitch). This results in relatively greater FKBP5 translation, unchecked by miR-320a. Overall, these results identify an important gene-miRNA interaction influencing chronic pain risk in vulnerable individuals and suggest that exogenous methods to achieve targeted reduction in poststress FKBP5 mRNA expression may constitute useful therapeutic strategies.SIGNIFICANCE STATEMENTFKBP5 is a critical regulator of the stress response. Previous studies have shown that dysregulation of the expression of this gene plays a role in the pathogenesis of chronic pain development as well as a number of comorbid neuropsychiatric disorders. In the current study, we identified a functional allele (rs3800373) in the 3'UTR of FKBP5 that influences vulnerability to chronic post-traumatic pain in two ethnic cohorts. Using multiple complementary experimental approaches, we show that the FKBP5 rs3800373 minor allele alters the secondary structure of FKBP5 mRNA, decreasing the binding of a stress- and pain-associated microRNA, miR-320a. This results in relatively greater FKBP5 translation, unchecked by miR-320a, increasing glucocorticoid resistance and increasing vulnerability to post-traumatic pain.

Multiple conformations are a conserved and regulatory feature of the RB1 5' UTR.

Folding to a well-defined conformation is essential for the function of structured ribonucleic acids (RNAs) like the ribosome and tRNA. Structured elements in the untranslated regions (UTRs) of specific messenger RNAs (mRNAs) are known to control expression. The importance of unstructured regions adopting multiple conformations, however, is still poorly understood. High-resolution SHAPE-directed Boltzmann suboptimal sampling of the Homo sapiens Retinoblastoma 1 (RB1) 5' UTR yields three distinct conformations compatible with the experimental data. Private single nucleotide variants (SNVs) identified in two patients with retinoblastoma each collapse the structural ensemble to a single but distinct well-defined conformation. The RB1 5' UTRs from Bos taurus (cow) and Trichechus manatus latirostris (manatee) are divergent in sequence from H. sapiens (human) yet maintain structural compatibility with high-probability base pairs. SHAPE chemical probing of the cow and manatee RB1 5' UTRs reveals that they also adopt multiple conformations. Luciferase reporter assays reveal that 5' UTR mutations alter RB1 expression. In a traditional model of disease, causative SNVs disrupt a key structural element in the RNA. For the subset of patients with heritable retinoblastoma-associated SNVs in the RB1 5' UTR, the absence of multiple structures is likely causative of the cancer. Our data therefore suggest that selective pressure will favor multiple conformations in eukaryotic UTRs to regulate expression.

Emergence of metastable state dynamics in interconnected cortical networks with propagation delays.

The importance of the large number of thin-diameter and unmyelinated axons that connect different cortical areas is unknown. The pronounced propagation delays in these axons may prevent synchronization of cortical networks and therefore hinder efficient information integration and processing. Yet, such global information integration across cortical areas is vital for higher cognitive function. We hypothesized that delays in communication between cortical areas can disrupt synchronization and therefore enhance the set of activity trajectories and computations interconnected networks can perform. To evaluate this hypothesis, we studied the effect of long-range cortical projections with propagation delays in interconnected large-scale cortical networks that exhibited spontaneous rhythmic activity. Long-range connections with delays caused the emergence of metastable, spatio-temporally distinct activity states between which the networks spontaneously transitioned. Interestingly, the observed activity patterns correspond to macroscopic network dynamics such as globally synchronized activity, propagating wave fronts, and spiral waves that have been previously observed in neurophysiological recordings from humans and animal models. Transient perturbations with simulated transcranial alternating current stimulation (tACS) confirmed the multistability of the interconnected networks by switching the networks between these metastable states. Our model thus proposes that slower long-range connections enrich the landscape of activity states and represent a parsimonious mechanism for the emergence of multistability in cortical networks. These results further provide a mechanistic link between the known deficits in connectivity and cortical state dynamics in neuropsychiatric illnesses such as schizophrenia and autism, as well as suggest non-invasive brain stimulation as an effective treatment for these illnesses.

Metazoan innovation: from aromatic amino acids to extracellular signaling.

Tyrosine depletion in metazoan proteins was recently explained to be due to the appearance of tyrosine kinases in Metazoa. Here, we present a complementary explanation for the depletion of tyrosine, stating the importance of tyrosine in signaling not only as a phosphorylation target but also as a precursor for catecholamines and hormones. Molecules (dopamine, norepinephrine, and epinephrine, and to a lesser extent serotonin and melatonin) critical to metazoan multicellular signaling are also greatly dependent on a supply of tyrosine. These signaling molecules are synthesized in two highly linked pathways specific to metazoans. In addition, the shikimate pathway that non-metazoans use to synthesize the aromatic amino acids is not present in metazoans. These important pathway changes have occurred between Metazoa and other eukaryotes, causing significant changes to tyrosine metabolism and rendering tyrosine crucial for extracellular signaling. In addition, the evolutionary and functional linkage between these two pathways and the resulting implications for neuropathology are discussed.

Transcending the prediction paradigm: novel applications of SHAPE to RNA function and evolution.

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) provides information on RNA structure at single-nucleotide resolution. It is most often used in conjunction with RNA secondary structure prediction algorithms as a probabilistic or thermodynamic restraint. With the recent advent of ultra-high-throughput approaches for collecting SHAPE data, the applications of this technology are extending beyond structure prediction. In this review, we discuss recent applications of SHAPE data in the transcriptomic context and how this new experimental paradigm is changing our understanding of these experiments and RNA folding in general. SHAPE experiments probe both the secondary and tertiary structure of an RNA, suggesting that model-free approaches for within and comparative RNA structure analysis can provide significant structural insight without the need for a full structural model. New methods incorporating SHAPE at different nucleotide resolutions are required to parse these transcriptomic data sets to transcend secondary structure modeling with global structural metrics. These 'multiscale' approaches provide deeper insights into RNA global structure, evolution, and function in the cell. WIREs RNA 2017, 8:e1374. doi: 10.1002/wrna.1374 For further resources related to this article, please visit the WIREs website.

Sensorimotor behavioral tests for use in a juvenile rat model of traumatic brain injury: assessment of sex differences.

Modeling juvenile traumatic brain injury (TBI) in rodents presents several unique challenges compared to adult TBI, one of which is selecting appropriate sensorimotor behavioral tasks that enable the assessment of the extent of injury and recovery over time in developing animals. To address this challenge, we performed a comparison of common sensorimotor tests in Long-Evans rats of various sizes and developmental stages (postnatal days 16-45, 35-190 g). Tests were compared and selected for their developmental appropriateness, scalability for growth, pre-training requirements, and throughput capability. Sex differences in response to TBI were also assessed. Grid walk, automated gait analysis, rotarod, beam walk, spontaneous forelimb elevation test, and measurement of motor activity using the force-plate actometer were evaluated. Grid walk, gait analysis, and rotarod failed to meet one or more of the evaluation criteria. Beam walk, spontaneous forelimb elevation test, and measurement of motor activity using the force-plate actometer satisfied all criteria and were capable of detecting motor abnormalities in rats subjected to controlled cortical impact on postnatal day 17. No sex differences were detected in the acute effects of TBI or functional recovery during the 28 days after injury using these tests. This demonstrates the utility of these tests for the evaluation of sensorimotor function in studies using rat models of pediatric TBI, and suggests that pre-pubertal males and females respond similarly to TBI with respect to sensorimotor outcomes.