Intramolecular Phenotypic Capacitance in a Modular RNA Molecule

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA’s ancient role in life suggests that phenotypic capacitance may have influenced evolution since life’s origins

Telomere Estimates by qPCR in American Kestrel Birds

Telomeres are highly conserved repeated sequences found at the ends of linear chromosomes. Measuring telomere length has been used to estimate the ages of individuals in several species. The research presented here focuses on developing telomere length estimates in the American Kestrel (Falco sparverius), using Quantitative Polymerase Chain Reaction (qPCR). The verification of a primer pair that can amplify a reference gene and serve to normalize the telomere qPCR data is vitally important. The gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was preferably chosen because it occurs in a single copy in most birds. However, the Kestrel genome has not been sequenced making primer design challenging. We used a bioinformatics approach to find regions of the GAPDH gene that are conserved between chicken and several species of raptors. We also checked several published GAPDH primers reported in the literature. Here, we report the sequence and efficiency of qPCR primers that can be used for telomere length studies in the American Kestral

Genotype Network Intersections Promote Evolutionary Innovation

Evolutionary innovations are qualitatively novel traits that emerge through evolution and increase biodiversity. The genetic mechanisms of innovation remain poorly understood. A systems view of innovation requires the analysis of genotype networks—the vast networks of genetic variants that produce the same phenotype. Innovations can occur at the intersection of two different genotype networks. However, the experimental characterization of genotype networks has been hindered by the vast number of genetic variants that need to be functionally analyzed. Here, we use high-throughput sequencing to study the fitness landscape at the intersection of the genotype networks of two catalytic RNA molecules (ribozymes). We determined the ability of numerous neighboring RNA sequences to catalyze two different chemical reactions, and we use these data as a proxy for a genotype to fitness map where two functions come in close proximity. We find extensive functional overlap, and numerous genotypes can catalyze both functions. We demonstrate through evolutionary simulations that these numerous points of intersection facilitate the discovery of a new function. However, the rate of adaptation of the new function depends upon the local ruggedness around the starting location in the genotype network. As a consequence, one direction of adaptation is more rapid than the other. We find that periods of neutral evolution increase rates of adaptation to the new function by allowing populations to spread out in their genotype network. Our study reveals the properties of a fitness landscape where genotype networks intersect and the consequences for evolutionary innovations. Our results suggest that historic innovations in natural systems may have been facilitated by overlapping genotype networks

RNA Sequence to Structure Analysis from Comprehensive Pairwise Mutagenesis of Multiple Self-Cleaving Ribozymes

Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes

Ribonucleoprotein Purification and Characterization Using RNA Mango

The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ42 Oligomers and Protect Synapses.

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Fibroblasts derived from oesophageal adenocarcinoma differ in DNA methylation profile from normal oesophageal fibroblasts

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Oesophageal adenocarcinoma (OAC) is increasing in incidence and has a poor prognosis. Tumour derived fibroblasts (TDFs) differ functionally from normal fibroblasts (NDFs), and play a pivotal role in cancer. Many of the differences persist through subculture. We measured the DNA methylation profiles of 10 TDFs from OAC with 12 NDF from normal oesophageal mucosa using Infinium HumanMethylation450 Beadchips and found they differed in multidimensional scaling analysis. We identified 4,856 differentially methylated CpGs (DMCs, adjusted p  0.15), of which 3,243 (66.8%) were hypomethylated in TDFs compared to NDFs. Hypermethylated DMCs were enriched at transcription start sites (TSSs) and in CpG islands, and depleted in transcriptional enhancers. Gene ontology analysis of genes with DMCs at TSSs revealed an enrichment of genes involved in development, morphogenesis, migration, adhesion, regulation of processes and response to stimuli. Alpha-smooth muscle actin (α-SMA) is a marker of activated fibroblasts and a poor prognostic indicator in OAC. Hypomethylated DMCs were observed at the TSS of transcript variant 2 of α-SMA, which correlated with an increase in α-SMA protein expression. These data suggest that DNA methylation may contribute to the maintenance of the TDF phenotype

Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity During \u3ci\u3ein vitro\u3c/i\u3e Expansion

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p \u3c 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p \u3c 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging