Native RNA G quadruplex Immunoprecipitation (rG4IP) from mammalian cells

G quadruplex structures play an important role in regulating DNA replication and transcription and mRNA translation. Although there are several techniques that can determine its formation in vitro, the study of RNA G quadruplexes in vivo is not simple. In the current protocol, we describe an optimized technique (RNA G quadruplex immunoprecipitation [rG4IP]) to selectively pull down native cytoplasmic RNAs containing G quadruplex structures in mammalian cells. We also use a bicistronic plasmid to confirm and pinpoint the structure location. For complete details on the use and execution of this protocol, please refer to Surani et al. (2022)

AGAP2: modulating TGFβ1-signalling in the regulation of liver fibrosis

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-β1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-β1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFβ1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis

Implications of differential Transcription Start Site selection on CML and prostate cancer cell protein expression

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukaemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5’ UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumour cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role on protein expression and should not be ignored

Role of AGAP2 in the profibrogenic effects induced by TGFβ in LX-2 hepatic stellate cells

Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor β 1 (TGFβ1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFβ1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFβ1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFβ1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFβ1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFβ1 induced AGAP2 promoter activation and its protein expression in LX-2. In addition, AGAP2 silencing affected TGFβ1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFβ1-induced increase of collagen-I protein levels, while its over-expression enhanced collagen I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFβ1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFβ1 signalling, contributing to the progression of hepatic fibrosis

SP1 and RARα regulate AGAP2 expression in cancer

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is considered a proto-oncogene, but not much is known about AGAP2 gene expression regulation. To get some insight into this process, AGAP2 proximal promoter was cloned and characterised using reporter assays. We have identified SP1 as a transcription factor bound to AGAP2 promoter and required for AGAP2 expression in two different types of cancer cells (KU812, a chronic myeloid leukaemia cell line; and DU145, a prostate cancer cell line): silencing SP1 decreased AGAP2 protein levels. We have also found that all-trans retinoic acid (ATRA) treatment increased AGAP2 protein levels in both cell lines whilst curcumin treatment reduced ATRA-mediated AGAP2 increase. Furthermore, chromatin immunoprecipitation studies revealed the presence of RARα, RXRα and the lysine acetyl transferase PCAF in AGAP2 promoter. Our results provide a novel understanding of AGAP2 expression regulation that could be beneficial to those patients with cancers where AGAP2 is overexpressed

BCR-ABL induces the expression of Skp2 through the PI3K pathway to promote p27Kip1 degradation and proliferation of chronic myelogenous leukemia cells

Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G(1) cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27(Kip1) and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias

Low exposure long-baseline neutrino oscillation sensitivity of the DUNE experiment

The Deep Underground Neutrino Experiment (DUNE) will produce world-leading neutrino oscillation measurements over the lifetime of the experiment. In this work, we explore DUNE's sensitivity to observe charge-parity violation (CPV) in the neutrino sector, and to resolve the mass ordering, for exposures of up to 100 kiloton-megawatt-years (kt-MW-yr). The analysis includes detailed uncertainties on the flux prediction, the neutrino interaction model, and detector effects. We demonstrate that DUNE will be able to unambiguously resolve the neutrino mass ordering at a 3$\sigma$ (5$\sigma$) level, with a 66 (100) kt-MW-yr far detector exposure, and has the ability to make strong statements at significantly shorter exposures depending on the true value of other oscillation parameters. We also show that DUNE has the potential to make a robust measurement of CPV at a 3$\sigma$ level with a 100 kt-MW-yr exposure for the maximally CP-violating values \delta_{\rm CP}} = \pm\pi/2. Additionally, the dependence of DUNE's sensitivity on the exposure taken in neutrino-enhanced and antineutrino-enhanced running is discussed. An equal fraction of exposure taken in each beam mode is found to be close to optimal when considered over the entire space of interest

A Gaseous Argon-Based Near Detector to Enhance the Physics Capabilities of DUNE

This document presents the concept and physics case for a magnetized gaseous argon-based detector system (ND-GAr) for the Deep Underground Neutrino Experiment (DUNE) Near Detector. This detector system is required in order for DUNE to reach its full physics potential in the measurement of CP violation and in delivering precision measurements of oscillation parameters. In addition to its critical role in the long-baseline oscillation program, ND-GAr will extend the overall physics program of DUNE. The LBNF high-intensity proton beam will provide a large flux of neutrinos that is sampled by ND-GAr, enabling DUNE to discover new particles and search for new interactions and symmetries beyond those predicted in the Standard Model

Snowmass Neutrino Frontier: DUNE Physics Summary

The Deep Underground Neutrino Experiment (DUNE) is a next-generation long-baseline neutrino oscillation experiment with a primary physics goal of observing neutrino and antineutrino oscillation patterns to precisely measure the parameters governing long-baseline neutrino oscillation in a single experiment, and to test the three-flavor paradigm. DUNE's design has been developed by a large, international collaboration of scientists and engineers to have unique capability to measure neutrino oscillation as a function of energy in a broadband beam, to resolve degeneracy among oscillation parameters, and to control systematic uncertainty using the exquisite imaging capability of massive LArTPC far detector modules and an argon-based near detector. DUNE's neutrino oscillation measurements will unambiguously resolve the neutrino mass ordering and provide the sensitivity to discover CP violation in neutrinos for a wide range of possible values of δCP. DUNE is also uniquely sensitive to electron neutrinos from a galactic supernova burst, and to a broad range of physics beyond the Standard Model (BSM), including nucleon decays. DUNE is anticipated to begin collecting physics data with Phase I, an initial experiment configuration consisting of two far detector modules and a minimal suite of near detector components, with a 1.2 MW proton beam. To realize its extensive, world-leading physics potential requires the full scope of DUNE be completed in Phase II. The three Phase II upgrades are all necessary to achieve DUNE's physics goals: (1) addition of far detector modules three and four for a total FD fiducial mass of at least 40 kt, (2) upgrade of the proton beam power from 1.2 MW to 2.4 MW, and (3) replacement of the near detector's temporary muon spectrometer with a magnetized, high-pressure gaseous argon TPC and calorimeter