Genome-scale approaches for discovering novel nonconventional splicing substrates of the Ire1 nuclease

BACKGROUND: The unfolded protein response (UPR) allows intracellular feedback regulation that adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The signal from the ER lumen is transmitted by the ER-transmembrane kinase Ire1, which upon activation displays a site-specific endoribonuclease activity. Endonucleolytic cleavage of the intron from the HAC1 mRNA (encoding a UPR-specific transcription factor) is the first step in a nonconventional mRNA splicing pathway; the released exons are then joined by tRNA ligase. Because only the spliced mRNA is translated, splicing is the key regulatory step of the UPR. RESULTS: We developed methods to search for additional mRNA substrates of Ire1p in three independent lines of genome-wide analysis. These methods exploited the well characterized enzymology and genetics of the UPR and the yeast genome sequence in conjunction with microarray-based detection. Each method successfully identified HAC1 mRNA as a substrate according to three criteria: HAC1 mRNA is selectively cleaved in vitro by Ire1; the HAC1 mRNA sequence contains two predicted Ire1 cleavage sites; and HAC1 mRNA is selectively degraded in tRNA ligase mutant cells. CONCLUSION: Within the limits of detection, no other mRNA satisfies any of these criteria, suggesting that a unique nonconventional mRNA-processing mechanism has evolved solely for carrying out signal transduction between the ER and the nucleus. The approach described here, which combines biochemical and genetic 'fractionation' of mRNA with a novel application of cDNA microarrays, is generally applicable to the study of pathways in which RNA metabolism and alternative splicing have a regulatory role

ZEB2 and MEIS1 independently contribute to hematopoiesis via early hematopoietic enhancer activation

血球細胞分化に必要な新たな因子を同定. 京都大学プレスリリース. 2023-09-29.Delineating the dynamic transcriptional and epigenetic landscape regulating hematopoiesis. 京都大学プレスリリース. 2023-10-17.Cell differentiation is achieved by acquiring a cell type-specific transcriptional program and epigenetic landscape. While the cell type-specific patterning of enhancers has been shown to precede cell fate decisions, it remains unclear how regulators of these enhancers are induced to initiate cell specification and how they appropriately restrict cells that differentiate. Here, using embryonic stem cell–derived hematopoietic cell differentiation cultures, we show the activation of some hematopoietic enhancers during arterialization of hemogenic endothelium, a prerequisite for hematopoiesis. We further reveal that ZEB2, a factor involved in the transcriptional regulation of arterial endothelial cells, and a hematopoietic regulator MEIS1 are independently required for activating these enhancers. Concomitantly, ZEB2 or MEIS1 deficiency impaired hematopoietic cell development. These results suggest that multiple regulators expressed from an earlier developmental stage non-redundantly contribute to the establishment of hematopoietic enhancer landscape, thereby restricting cell differentiation despite the unrestricted expression of these regulators to hematopoietic cells

Petit-exposure at neutrino beamline (PEANUT)

The advantages of using nuclear emulsion as a particle detector are well known. The high resolution of emulsion has made it a medium of choice for a number of applications where the required spatial and angular resolution are paramount and its limitations due to the lack of timing information are less important. Emulsions are commonly used as cosmic ray detectors and have found applications in high energy experiments for detecting short lived particles such as charm, beauty and tau. The addition of electronic detectors to emulsion experiments solved the problem of the lack of timing information in the emulsion, but it was the development of automatic scanning machines that revolutionized the use of these hybrid detectors, making them capable of performing even in high rate environments. Most recently, The DONuT experiment (FNAL-E872), used a hybrid emulsion spectrometer to make the first direct observation of tau neutrino interactions [1]. The CNGS facility is being constructed to deliver a {nu}{sub {mu}} beam from the CERN SPS to the Gran Sasso Laboratory. Since it is believed that {nu}{sub {mu}} {leftrightarrow} {nu}{sub {tau}} oscillations explain the observed atmospheric {nu}{sub {mu}} deficit, the CNGS beam, coupled with a detector capable of observing {tau} appearance is an important experiment in the context of the world wide effort to determine the neutrino mass mixing matrix. The OPERA detector has been optimized to detect a significant sample of {nu}{sub {tau}} interactions by the subsequent observation of {tau} production and decay [2]. The OPERA target is a massive emulsion detector made in a sandwich structure of lead plates and layers of nuclear emulsion. For historical reasons this arrangement has been called an Emulsion Cloud Chamber or ECC. The ECC concept, which has many advantages over the use of bulk emulsion, has been used in the DONuT experiment. The ECC detector is capable of measuring all of the tracks, not due to nuclear fragments, coming from the primary neutrino interaction vertex, with their three dimensional slopes and momenta. It is also capable of electron identification with good e/{gamma} separation, due to its very fine segmentation. The OPERA ECC target modules are constructed as bricks of dimensions 12.5 x 10.0 x 7.5 cm{sup 3} in horizontal, vertical and along the beam axis. Each brick consists of series of 56 (1 mm thick) plates of passive material (lead or iron) alternated with emulsion films (43 {micro}m emulsion layer on both sides of a transparent 200 {micro}m thick plastic film). In preparation for OPERA we would like to expose the OPERA target modules to a beam of neutrinos. This will allow us to test many of our analysis procedures and techniques as well as to validate the simulation of neutrino interactions, both for the production of forward and backward particles. Although the HE (high energy) beam of NuMI would be a better match to the CNGS energy, data acquired with NuMI LE (low energy) beam would serve the same purpose, albeit more challenging. Given the high interaction rate from the NuMI beam, the test detector target mass can be kept low and additional detectors can easily be built around a small target. These measurements are not possible in the CNGS beam, since it has no short baseline hall

Simulating the Mammalian Blastocyst - Molecular and Mechanical Interactions Pattern the Embryo

Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v) and embryonic-abembryonic (eb-ab) axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1) The position determines gene expression, and (2) the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1) A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM). In this case the blastocoel simply acts as a static boundary. (2) The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial mechanical simulations with genetic networks to explain mammalian embryogenesis. Such a framework provides the means to test hypotheses in a controlled in silico environment