Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.Alternative splicing of pre-mRNA leads to the synthesis of multiple protein isoforms from a single gene. It is an important mechanism for regulating gene expression and may be utilized by 40 -60% of human genes (1-4). Thus, the estimated 25,000 to 30,000 genes of the human genome can generate a much larger number of proteins. Regulation of alternative splicing occurs in both a tissue-and development-specific manner, resulting in alterations in the structure and function of critical proteins. Altered splicing regulation can also be of widespread importance in the etiology of human disease (5-7).The protein 4.1 gene family serves as an excellent model for investigating the regulation of alternative splicing. The four genes that comprise the family (4 .1R, 4.1G, 4.1B, and 4.1N) display a remarkable array of highly regulated, tissue-specific splicing events. These alternative splicing events facilitate expression of distinct isoforms of 4.1 protein in cells of erythroid, epithelial, neural, and muscle origin (8 -14); thus, they provide opportunities for understanding the mechanisms that regulate alternative splicing in several different cell types. To date, mechanistic studies have focused predominantly on erythroid cells, in which 4.1R protein is a structural component of the erythrocyte plasma membrane and is important for structural integrity and stability of the membrane skeleton. In differentiating erythroid progenitor cells, a dramatic switch in pre-mRNA splicing result...
Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-␣-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACEmediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes.Ectodomain shedding is an essential phenomenon involved in the cleavage and release of cell membrane-bound molecules ranging from Alzheimer's amyloid precursor protein to angiotensin-converting enzyme (1). Shedding of ligand/receptor families is involved in diverse processes such as inflammation, hematopoiesis, and normal development (2, 3). Ectodomain shedding can be stimulated by protein kinase C activation and endotoxin (4,5). TACE 1 is a member of a disintegrin and metalloproteinase (ADAM) family, a group of unique zinc-binding transmembrane metalloproteinases (6 -8). TACE has been shown to mediate cleavage of TNF␣ as well as a variety of ectodomains including the TNF p75 receptor, L-selectin, and transforming growth factor-␣ (3). Despite its importance, the physiological regulation of TACE activity remains undefined. Endotoxin does not alter TACE transcription, steady-state mRNA levels, or the level of processed TACE at the cell surface (3). However, endotoxin can induce both nitric oxide (NO) and free radical production (9 -11). It has been shown that both hydrogen peroxide and NO can enhance macrophage production and the secretion of TNF␣ (12-16). However, the mechanism of this effect remains unclear. Many metalloproteinases can be activated by oxidation and dissociation of the cysteine thiol-zinc linkage from a latent enzymatic site, with this complex referred to as a "cysteine zinc switch" mechanism (17). TACE contains a consensus cysteine switch motif in the prodomain, and it has been shown previously that the cysteine in this portion of the molecule is required for the inhibition of TACE activity (18). In the present study, we tested the hypothesis that NO, a molecule produced in a variety of inflammatory conditions, regulates TACE activity and ectodomain shedding. EXPERIMENTAL PROCEDURES Chemicals-(Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]-diazen-, and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one were obtained from Alexis Biochemicals (San Diego, CA). Actinomycin D and oxyhemoglobin were from Sigma. Murine interferon-␥ was provided by Genentech (South San Francisco, CA). Escherichia coli LPS 026:B6 was from Difco. All other chemicals were purchased from Sigma.Cell Culture-Mono Mac 6 and Jurkat...
A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE 16 ) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model premRNAs showed that CE 16 can repress splicing of upstream introns, and that mutagenesis or replacement of CE 16 can relieve this inhibition. An af®nity selection assay with biotinylated CE 16 RNA demonstrated speci®c binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract signi®cantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-speci®c activation of the E16 splicing switch in concert with a dramatic and speci®c down-regulation of hnRNP A/B protein expression. These ®ndings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in premRNA splicing. Keywords: alternative splicing/exonic splicing silencer/ hnRNP A and B/protein 4.1R
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.