scholarly journals Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon–exon junctions

2000 ◽  
Vol 14 (9) ◽  
pp. 1098-1108 ◽  
Author(s):  
Hervé Le Hir ◽  
Melissa J. Moore ◽  
Lynne E. Maquat
Keyword(s):  
Direct Evidence ◽  
Protein Composition ◽  
Mrna Splicing ◽  
Core Component ◽  
Nonsense Mediated Decay ◽  
Exon Junctions ◽  
Stable Association ◽  
Gradient Fractionation

We provide direct evidence that pre-mRNA splicing alters mRNP protein composition. Using a novel in vitro cross-linking approach, we detected several proteins that associate with mRNA exon–exon junctions only as a consequence of splicing. Immunoprecipitation experiments suggested that these proteins are part of a tight complex around the junction. Two were identified as SRm160, a nuclear matrix-associated splicing coactivator, and hPrp8p, a core component of U5 snRNP and spliceosomes. Glycerol gradient fractionation showed that a subset of these proteins remain associated with mRNA after its release from the spliceosome. These results demonstrate that the spliceosome can leave behind signature proteins at exon–exon junctions. Such proteins could influence downstream metabolic events in vivo such as mRNA transport, translation, and nonsense-mediated decay.

scholarly journals Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma

NAR Cancer ◽  
2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Verónica Freire-Benéitez ◽  
Nicola Pomella ◽  
Thomas O Millner ◽  
Anaëlle A Dumas ◽  
Maria Victoria Niklison-Chirou ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Protein Composition ◽  
Mrna Splicing ◽  
Cholesterol Transport ◽  
Normal Brain ◽  
Regulatory Functions ◽  
Polycomb Repressive Complex 1 ◽  
Normal Brain Development

Abstract Glioblastoma (GBM) is the most common and aggressive intrinsic brain tumour in adults. Epigenetic mechanisms controlling normal brain development are often dysregulated in GBM. Among these, BMI1, a structural component of the Polycomb Repressive Complex 1 (PRC1), which promotes the H2AK119ub catalytic activity of Ring1B, is upregulated in GBM and its tumorigenic role has been shown in vitro and in vivo. Here, we have used protein and chromatin immunoprecipitation followed by mass spectrometry (MS) analysis to elucidate the protein composition of PRC1 in GBM and transcriptional silencing of defining interactors in primary patient-derived GIC lines to assess their functional impact on GBM biology. We identify novel regulatory functions in mRNA splicing and cholesterol transport which could represent novel targetable mechanisms in GBM.

TAMI-73. GLIOBLASTOMA CELL POPULATIONS WITH DISTINCT ONCOGENIC PROGRAMS RELEASE PODOPLANIN AS PROCOAGULANT EXTRACELLULAR VESICLES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi213-vi213
Author(s):  
Nadim Tawil ◽  
Rayhaan Bassawon ◽  
Brian Meehan ◽  
Laura Montermini ◽  
Ali Nehme ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Glioblastoma Cell ◽  
Activation Markers ◽  
Increased Risk ◽  
Gradient Fractionation ◽  
D Dimer

Abstract BACKGROUND Vascular anomalies, including thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of dysregulated cancer cell genome and epigenome. Up-regulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk of venous thromboembolism in glioblastoma patients. Thus, regulation of this platelet activating protein by transforming events and release from cancer cells is of considerable interest. AIMS I. Investigate the pattern of PDPN expression and characterize PDPN-expressing cellular populations in GBM. II. Evaluate the contribution of oncogenic drivers to PDPN expression in GBM models. III. Investigate the potential involvement of extracellular vesicles (EVs) as a mechanism for systemic dissemination of PDPN and tissue factor (TF). IV. Examine the role of PDPN in intratumoral and systemic thrombosis. METHODS Bioinformatics (single-cell and bulk transcriptome data mining), GBM cell lines and stem cell lines, xenograft models in mice, ELISA assays for PDPN and TF, platelet (PF4) and clotting activation markers (D-dimer), EV electron microscopy, density gradient fractionation, and nano-flow cytometry. RESULTS PDPN is expressed by distinct glioblastoma cell subpopulations (mesenchymal) and downregulated by oncogenic mutations of EGFR and IDH1 genes, via changes in chromatin modifications (EZH2) and DNA methylation, respectively. GBM cells exteriorize PDPN and/or TF as cargo of exosome-like EVs shed both in vitro and in vivo. Injection of glioma PDPN-EVs activates platelets. Increase of platelet activation (PF4) or coagulation markers (D-dimer) occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Co-expression of PDPN and TF by GBM cells cooperatively increases tumor microthrombosis. CONCLUSION Distinct cellular subsets drive multiple facets of GBM-associated thrombosis and may represent targets for diagnosis and intervention. We suggest that the preponderance of PDPN expression as a risk factor in glioblastoma and the involvement of platelets may merit investigating anti-platelets for potential inclusion in thrombosis management in GBM.

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

scholarly journals Reading and Function of a Histone Code Involved in Targeting Corepressor Complexes for Repression

2005 ◽  
Vol 25 (1) ◽  
pp. 324-335 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Youngsok Choi ◽  
Philip A. Cole ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hormone Receptors ◽  
Critical Role ◽  
Pericentric Heterochromatin ◽  
Histone Code ◽  
Stable Association ◽  
And Function

ABSTRACT A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

YY1 and RTCB in mouse uterine decidualization and embryo implantation

Reproduction ◽  
2021 ◽  
Author(s):  
Ran Li ◽  
Xiao-Tong Song ◽  
Si-Wei Guo ◽  
Na Zhao ◽  
Mei He ◽  
...  
Keyword(s):  
Early Pregnancy ◽  
Embryo Implantation ◽  
Mrna Splicing ◽  
Proximal Promoter ◽  
Mouse Uterus ◽  
Rna Ligase ◽  
Xbp1 Mrna ◽  
Transcription Factor Yy1

As a multifunctional transcription factor, YY1 regulates the expression of many genes essential for early embryonic development. RTCB is an RNA ligase that plays a role in tRNA maturation and Xbp1 mRNA splicing. YY1 can bind in vitro to the response element in the proximal promoter of Rtcb and regulate Rtcb promoter activity. However, the in vivo regulation and whether these two genes are involved in the mother-fetal dialogue during early pregnancy remain unclear. In this study, we validated that YY1 bound in vivo to the proximal promoter of Rtcb in mouse uterus of early pregnancy. Moreover, via building a variety of animal models, our study suggested that both YY1 and RTCB might play a role in mouse uterus decidualization and embryo implantation during early pregnancy.

scholarly journals Live View of Gonadotropin-Releasing Hormone Containing Neuron Migration

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 463-468 ◽  
Author(s):  
Elizabeth P. Bless ◽  
Heather J. Walker ◽  
Kwok W. Yu ◽  
J. Gabriel Knoll ◽  
Suzanne M. Moenter ◽  
...  
Keyword(s):  
Real Time ◽  
Direct Evidence ◽  
Gonadotropin Releasing Hormone ◽  
Releasing Hormone ◽  
Reproductive Axis ◽  
Gnrh Neurons ◽  
Migratory Route ◽  
The Brain

Neurons that synthesize GnRH control the reproductive axis and migrate over long distances and through different environments during development. Prior studies provided strong clues for the types of molecules encountered and movements expected along the migratory route. However, our studies provide the first real-time views of the behavior of GnRH neurons in the context of an in vitro preparation that maintains conditions comparable to those in vivo. The live views provide direct evidence of the changing behavior of GnRH neurons in their different environments, showing that GnRH neurons move with greater frequency and with more changes in direction after they enter the brain. Perturbations of guiding fibers distal to moving GnRH neurons in the nasal compartment influenced movement without detectable changes in the fibers in the immediate vicinity of moving GnRH neurons. This suggests that the use of fibers by GnRH neurons for guidance may entail selective signaling in addition to mechanical guidance. These studies establish a model to evaluate the influences of specific molecules that are important for their migration.

scholarly journals Alterations of protein composition along the rostro-caudal axis after spinal cord injury: proteomic, in vitro and in vivo analyses

2014 ◽  
Vol 8 ◽  
Author(s):  
Dasa Cizkova ◽  
Françoise Le Marrec-Croq ◽  
Julien Franck ◽  
Lucia Slovinska ◽  
Ivana Grulova ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Protein Composition ◽  
Cord Injury

scholarly journals Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation.

1989 ◽  
Vol 109 (4) ◽  
pp. 1571-1579 ◽  
Author(s):  
J H Hartwig ◽  
K A Chambers ◽  
K L Hopcia ◽  
D J Kwiatkowski
Keyword(s):  
Platelet Activation ◽  
Direct Evidence ◽  
Human Leukocyte ◽  
Cell Types ◽  
Response To Treatment ◽  
Membrane Association ◽  
Actin Monomer ◽  
High Affinity

Profilin is a conserved, widely distributed actin monomer binding protein found in eukaryotic cells. Mammalian profilin reversibly sequesters actin monomers in a high affinity profilactin complex. In vitro, the complex is dissociated in response to treatment with the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol 4,5-bisphosphate. Here, we demonstrate the ultrastructural immunolocalization of profilin in human leukocytes and platelets. In both cell types, a significant fraction of profilin is found associated with regions of cell membrane devoid of actin filaments and other discernible structures. After platelet activation, the membrane association of profilin reversibly increases. This study represents the first direct evidence for an interaction between profilin and phospholipids in vivo.

scholarly journals MukEF Is Required for Stable Association of MukB with the Chromosome

2007 ◽  
Vol 189 (19) ◽  
pp. 7062-7068 ◽  
Author(s):  
Weifeng She ◽  
Qinhong Wang ◽  
Elena A. Mordukhova ◽  
Valentin V. Rybenkov
Keyword(s):  
Fluorescent Protein ◽  
Cell Length ◽  
Stable Complex ◽  
Macromolecular Assembly ◽  
Stable Association ◽  
Structural Maintenance Of Chromosome ◽  
Green Fluorescent

ABSTRACT MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.

A Key Role of Adenosine Diphosphate in the Irreversible Platelet Aggregation Induced by the PAR1-Activating Peptide Through the Late Activation of Phosphoinositide 3-Kinase

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4156-4165 ◽  
Author(s):  
Catherine Trumel ◽  
Bernard Payrastre ◽  
Monique Plantavid ◽  
Béatrice Hechler ◽  
Cécile Viala ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Kinase Inhibitors ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Activation Process ◽  
Irreversible Aggregation ◽  
Stable Association ◽  
Phosphoinositide 3 Kinase

Abstract Although adenosine diphosphate (ADP), per se, is a weak platelet agonist, its role as a crucial cofactor in human blood platelet functions has now been clearly demonstrated in vitro and in vivo. The molecular basis of the ADP-induced platelet activation is starting to be understood since the discovery that 2 separate P2 purinergic receptors may be involved simultaneously in the activation process. However, little is known about how ADP plays its role as a cofactor in platelet activation and which signaling pathway initiated by a specific agonist can be modulated by the released ADP. To investigate these points, we took advantage of a model of platelet activation through the thrombin receptor PAR1 in which both ADP scavengers and phosphoinositide 3-kinase (PI 3-kinase) inhibitors have been shown to transform the classical irreversible aggregation into a reversible one. We have observed that, among the different PI 3-kinase products, the accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] was dramatically and specifically attenuated when ADP was removed by apyrase treatment. A comparison between the effects of PI 3-kinase inhibitors and apyrase strongly suggest that the late, ADP-dependent, PtdIns(3,4)P2accumulation is necessary for PAR1-induced irreversible aggregation. Using selective antagonists, we found that the effect of ADP was due to the ADP receptor coupled to inhibition of adenylyl cyclase. Finally, we found that both ADP and PI 3-kinase play an important role in PAR1-dependent reorganization of the cytoskeleton through a control of myosin heavy chain translocation and the stable association of signaling complexes with the actin cytoskeleton.

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