Quantitative analysis of competition in post-transcriptional regulation reveals a novel signature in target expression variation

When small RNAs are loaded onto Argonaute proteins they can form the RNA-induced silencing complexes (RISCs), which mediate RNA interference. RISC-formation is dependent on a shared pool of Argonaute proteins and RISC loading factors, and is thus susceptible to competition among small RNAs for loading. We present a mathematical model that aims to understand how small RNA competition for the PTR resources affects target gene repression. We discuss that small RNA activity is limited by RISC-formation, RISC-degradation and the availability of Argonautes. Together, these observations explain a number of PTR saturation effects encountered experimentally. We show that different competition conditions for RISC-loading result in different signatures of PTR activity determined also by the amount of RISC-recycling taking place. In particular, we find that the small RNAs less efficient at RISC-formation, using fewer resources of the PTR pathway, can perform in the low RISC-recycling range equally well as their more effective counterparts. Additionally, we predict a novel signature of PTR in target expression levels. Under conditions of low RISC-loading efficiency and high RISC-recycling, the variation in target levels increases linearly with the target transcription rate. Furthermore, we show that RISC-recycling determines the effect that Argonaute scarcity conditions have on target expression variation. Our observations taken together offer a framework of predictions which can be used in order to infer from experimental data the particular characteristics of underlying PTR activity.Comment: 23 pages, 3 Figures, accepted for publication to the Biophysical Journa

Epigenetic dynamics of monocyte-to-macrophage differentiation

Background Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. Results Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. Conclusions In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation

Phonon-mediated tuning of instabilities in the Hubbard model at half-filling

We obtain the phase diagram of the half-filled two-dimensional Hubbard model on a square lattice in the presence of Einstein phonons. We find that the interplay between the instantaneous electron-electron repulsion and electron-phonon interaction leads to new phases. In particular, a dx2−y2-wave superconducting phase emerges when both anisotropic phonons and repulsive Hubbard interaction are present. For large electron-phonon couplings, charge-density-wave and s-wave superconducting regions also appear in the phase diagram, and the widths of these regions are strongly dependent on the phonon frequency, indicating that retardation effects play an important role. Since at half filling the Fermi surface is nested, a spin-density wave is recovered when the repulsive interaction dominates. We employ a functional multiscale renormalization-group method [Tsai et al., Phys. Rev. B 72, 054531 (2005)] that includes both electron-electron and electron-phonon interactions, and take retardation effects fully into account

How epigenetic mutations can affect genetic evolution: Model and mechanism

We hypothesize that heritable epigenetic changes can affect rates of fitness increase as well as patterns of genotypic and phenotypic change during adaptation. In particular, we suggest that when natural selection acts on pure epigenetic variation in addition to genetic variation, populations adapt faster, and adaptive phenotypes can arise before any genetic changes. This may make it difficult to reconcile the timing of adaptive events detected using conventional population genetics tools based on DNA sequence data with environmental drivers of adaptation, such as changes in climate. Epigenetic modifications are frequently associated with somatic cell differentiation, but recently epigenetic changes have been found that can be transmitted over many generations. Here, we show how the interplay of these heritable epigenetic changes with genetic changes can affect adaptive evolution, and how epigenetic changes affect the signature of selection in the genetic record

Characterization of transcription termination associated-RNAs (TTSa-RNAs): new insights into their biogenesis, tailing and expression in primary tumors

Next-generation sequencing has uncovered novel classes of small RNAs(sRNAs)in eukaryotes,in 30addition to the well-known miRNAs, siRNAs and piRNAs.In particular, sRNAspeciesarise from 31transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a 32detailed characterization of these new classes of sRNAs is still lacking. 33Here we present a comprehensive study of sRNAs derived from TTSsof expressed genes (TTSa-RNAs) 34in human cell lines and primary tissues.Taking advantage of sRNA-sequencing, we show that TTSa-35RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2 and their 36biogenesis does not requireDICER andAGO2 endonucleolytic activity. TTSa-RNAsdisplay a strong37bias against a G residue in the first position at 5' end, a knownfeature of AGO-bound sRNAs, and a 38peculiar oligoA tail at 3’ end.AGO-bound TTSa-RNAs derive from genes involved in cell cycle 39progression regulationand DNA integrity checkpoints.Finally, we provide evidence that TTSa-RNAs 40can be detected by sRNA-Seq in primary human tissue and their expression increases in tumor samples 41as compared to non-tumor tissues, suggesting that in the future TTSa-RNAs might be explored as 42biomarker for diagnosis or prognosis of human malignancie

The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway

The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 50 leader and 30 trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures