Dynamic Energy Landscapes of Riboswitches Help Interpret Conformational Rearrangements and Function

Riboswitches are RNAs that modulate gene expression by ligand-induced conformational changes. However, the way in which sequence dictates alternative folding pathways of gene regulation remains unclear. In this study, we compute energy landscapes, which describe the accessible secondary structures for a range of sequence lengths, to analyze the transcriptional process as a given sequence elongates to full length. In line with experimental evidence, we find that most riboswitch landscapes can be characterized by three broad classes as a function of sequence length in terms of the distribution and barrier type of the conformational clusters: low-barrier landscape with an ensemble of different conformations in equilibrium before encountering a substrate; barrier-free landscape in which a direct, dominant “downhill” pathway to the minimum free energy structure is apparent; and a barrier-dominated landscape with two isolated conformational states, each associated with a different biological function. Sharing concepts with the “new view” of protein folding energy landscapes, we term the three sequence ranges above as the sensing, downhill folding, and functional windows, respectively. We find that these energy landscape patterns are conserved in various riboswitch classes, though the order of the windows may vary. In fact, the order of the three windows suggests either kinetic or thermodynamic control of ligand binding. These findings help understand riboswitch structure/function relationships and open new avenues to riboswitch design

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

<p>Abstract</p> <p>Background</p> <p>Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD). One hypothesis is that amyloid beta (Aβ) peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD.</p> <p>Methods</p> <p>Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis.</p> <p>Results</p> <p>Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals.</p> <p>Conclusions</p> <p>These data suggest that amyloid dependent microgliosis may be Src kinase dependent <it>in vitro</it> and <it>in vivo.</it> This study defines a role for Src kinase in the microgliosis characteristic of diseased brains and suggests that particular tyrosine kinase inhibition may be a valid anti-inflammatory approach to disease. Dasatinib is an FDA-approved drug for treating chronic myeloid leukemia cancer with a reported ability to cross the blood-brain barrier. Therefore, this suggests a novel use for this drug as well as similar acting molecules.</p

RNA editing in mitochondrial trans-introns is required for splicing

In plant mitochondria, gene expression of translatable mRNAs is a complex process with two critical steps, RNA editing and splicing. We studied the role of RNA editing on non-coding regions of the mat-r-nad1e-nad5c transcript from wheat mitochondria. This RNA contains two trans-introns, 3'-nad1-I4 and 3'-nad5-I2, involved in different trans-splicing events, ensuring the association of nad1d-nad1e and nad5b-nad5c exons from nad1 and nad5 mRNAs respectively. The C-to-U editing changes studied here affect homologous positions on 3'-nad1-I4 and 3'-nad5-I2. It is proposed that these base changes are necessary to place an Adenosine residue in a bulging conformation characteristic of domain VI (D6) from group II introns. In this work, we investigated the role of RNA editing events on 3'-nad1-I4 and 3'-nad5-I2 in the trans-splicing process using in vivo and in organello approaches. When the branched intermediates formed during the splicing process were analyzed, the C residues from D6 intron domains from 3'-nad1-I4 and 3'-nad5-I2 were found changed to U, suggesting that RNA editing of these residues could be mandatory for splicing. This assumption was tested by expressing recombinant mat-r-nad1e transgenes introduced into mitochondria by electroporation. Mutation of the editing target residue dramatically affected trans-splicing. Interestingly, the exon joining efficiency was not recovered by compensatory mutations, suggesting that the role of RNA editing is not confined to the restoration of the secondary structure of domain D6 of the intron. Our results strongly support the hypothesis that RNA editing in trans-introns precedes maturation, and is required for the splicing reaction. In addition, this is the first report using an in organello approach to study the trans-splicing process, opening the way to future studies of this peculiar mechanism

Drawing and Editing the Secondary Structure(s) of RNA.

International audienceSecondary structure diagrams are essential, in RNA biology, to communicate functional hypotheses and summarize structural data, and communicate them visually as drafts or finalized publication-ready figures. While many tools are currently available to automate the production of such diagrams, their capacities are usually partial, making it hard for a user to decide which to use in a given context. In this chapter, we guide the reader through the steps involved in the production of expressive publication-quality illustrations featuring the RNA secondary structure. We present major existing representations and layouts, and give precise instructions to produce them using available free software, including jViz.RNA, the PseudoViewer, RILogo, R-chie, RNAplot, R2R, and VARNA. We describe the file formats and structural descriptions accepted by popular RNA visualization tools. We also provide command lines and Python scripts to ease the user's access to advanced features. Finally, we discuss and illustrate alternative approaches to visualize the secondary structure in the presence of probing data, pseudoknots, RNA-RNA interactions, and comparative data