scholarly journals A Sequence-Specific RNA-Binding Protein Complements Apobec-1 To Edit Apolipoprotein B mRNA

1998 ◽  
Vol 18 (8) ◽  
pp. 4426-4432 ◽  
Author(s):  
Anuradha Mehta ◽  
Donna M. Driscoll
Keyword(s):  
Apolipoprotein B ◽  
Rna Binding ◽  
Protein A ◽  
Antisense Strand ◽  
Wild Type ◽  
Triple Mutant ◽  
Apo B ◽  
Editing Enzyme ◽  
Binding Subunit

ABSTRACT The editing of apolipoprotein B (apo-B) mRNA involves the site-specific deamination of cytidine to uracil. The specificity of editing is conferred by an 11-nucleotide mooring sequence located downstream from the editing site. Apobec-1, the catalytic subunit of the editing enzyme, requires additional proteins to edit apo-B mRNA in vitro, but the function of these additional factors, known as complementing activity, is not known. Using RNA affinity chromatography, we show that the complementing activity binds to a 280-nucleotide apo-B RNA in the absence of apobec-1. The activity did not bind to the antisense strand or to an RNA with three mutations in the mooring sequence. The eluate from the wild-type RNA column contained a 65-kDa protein that UV cross-linked to apo-B mRNA but not to the triple-mutant RNA. This protein was not detected in the eluates from the mutant or the antisense RNA columns. Introduction of the mooring sequence into luciferase RNA induced cross-linking of the 65-kDa protein. A 65-kDa protein that interacted with apobec-1 was also detected by far-Western analysis in the eluate from the wild-type RNA column but not from the mutant RNA column. For purification, proteins were precleared on the mutant RNA column prior to chromatography on the wild-type RNA column. Silver staining of the affinity-purified fraction detected a single prominent protein of 65 kDa. Our results suggest that the complementing activity may function as the RNA-binding subunit of the holoenzyme.

scholarly journals Molecular Cloning of Apobec-1 Complementation Factor, a Novel RNA-Binding Protein Involved in the Editing of Apolipoprotein B mRNA

2000 ◽  
Vol 20 (5) ◽  
pp. 1846-1854 ◽  
Author(s):  
Anuradha Mehta ◽  
Michael T. Kinter ◽  
Nicholas E. Sherman ◽  
Donna M. Driscoll
Keyword(s):  
Molecular Cloning ◽  
Apolipoprotein B ◽  
Rna Binding ◽  
Peptide Sequence ◽  
Cross Linking ◽  
Sequence Information ◽  
Apo B ◽  
Cytidine Deaminase Activity ◽  
Editing Enzyme

ABSTRACT The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.

Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA

1993 ◽  
Vol 13 (12) ◽  
pp. 7288-7294
Author(s):  
D M Driscoll ◽  
S Lakhe-Reddy ◽  
L M Oleksa ◽  
D Martinez
Keyword(s):  
Rna Editing ◽  
Apolipoprotein B ◽  
Point Mutations ◽  
Upstream Region ◽  
Wild Type ◽  
Cross Link ◽  
Apo B ◽  
Optimal Distance ◽  
Luciferase Mrna

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.

scholarly journals Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA.

1993 ◽  
Vol 13 (12) ◽  
pp. 7288-7294 ◽  
Author(s):  
D M Driscoll ◽  
S Lakhe-Reddy ◽  
L M Oleksa ◽  
D Martinez
Keyword(s):  
Rna Editing ◽  
Apolipoprotein B ◽  
Point Mutations ◽  
Upstream Region ◽  
Wild Type ◽  
Cross Link ◽  
Apo B ◽  
Optimal Distance ◽  
Luciferase Mrna

An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.

scholarly journals Regulatable liver expression of the rabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in mice lacking endogenous APOBEC-1 leads to aberrant hyperediting

2003 ◽  
Vol 369 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Martin HERSBERGER ◽  
Susannah PATARROYO-WHITE ◽  
Xiaobing QIAN ◽  
Kay S. ARNOLD ◽  
Lucia ROHRER ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Time Course ◽  
Single Gene ◽  
Wild Type ◽  
Mrna Editing ◽  
Apo B ◽  
Transgenic Expression ◽  
Apob Mrna ◽  
Time Course Experiment ◽  
Editing Enzyme

Apolipoprotein (apo) B mRNA editing is the deamination of C6666 to uridine, which results in translation of the apoB-48 protein instead of the genomically encoded apoB-100. ApoB-48-containing lipoproteins are cleared more rapidly from plasma and are less atherogenic than apoB-100-containing low-density lipoproteins (LDLs). In humans, the intestine predominantly produces apoB-48 whereas the liver secretes apoB-100 only. To evaluate a potential therapeutic use for liver-induced apoB mRNA editing in humans, we investigated the efficiency and safety of transgenic expression of apoB mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in the absence of endogenous editing in the mouse model. Here we show that regulatable tetO-mediated APOBEC-1 expression in the livers of gene-targeted mice lacking endogenous APOBEC-1 results in 30% apoB mRNA editing. In a time-course experiment, the expression of tetO-APOBEC-1 mRNA was suppressed within 2 days after mice were fed doxycycline and apoB mRNA editing and apoB-48 formation were suppressed within 4 days. However, tetO-APOBEC-1 expression resulted in regulatable aberrant hyperediting of several cytidines downstream of C6666 in apoB mRNA and in novel APOBEC-1 target 1 (NAT1) mRNA. Several of the cytidines in apoB mRNA were hyperedited to a level similar to that of C6666, although editing at C6666 was lower than that in wild-type mice. These results demonstrate that even moderate APOBEC-1 expression can lead to hyperediting, limiting the single-gene approach for gene therapy with APOBEC-1.

Abstract 229: TNF Receptor 1 Signaling Is Critically Involved in Mediating Angiotensin II-Induced Cardiac Fibrosis and Dysfunction

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Sandra B Haudek ◽  
Jeff Crawford ◽  
Erin Reineke ◽  
Alberto A Allegre ◽  
George E Taffet ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Fibrosis ◽  
Smooth Muscle Actin ◽  
Protein A ◽  
Ang Ii ◽  
Wild Type ◽  
Monocyte Chemoattractant Protein 1 ◽  
Reactive Fibrosis

Angiotensin-II (Ang-II) plays a key role in the development of cardiomyopathies, as it is associated with many conditions involving heart failure and pathologic hypertrophy. Using a murine model of Ang-II infusion, we found that Ang-II induced the synthesis of monocyte chemoattractant protein 1 (MCP-1) that mediated the uptake of CD34 + /CD45 + monocytic cells into the heart. These precursor cells differentiated into collagen-producing fibroblasts and were responsible for the Ang-II-induced development of reactive fibrosis. Preliminary in vitro data using our monocyte-to-fibroblast differentiation model, suggested that Ang-II required the presence of TNF to induce fibroblast maturation from monocytes. In vivo, they indicated that in mice deficient of both TNF receptors (TNFR1 and TNFR2), Ang-II-induced fibrosis was absent. We now assessed the hypothesis that specific TNFR1 signaling is necessary for Ang-II-mediated cardiac fibrosis. Mice deficient in either TNFR1 (TNFR1-KO) or TNFR2 (TNFR2-KO) were subjected to continuous infusion of Ang-II for 1 to 6 weeks (n=6-8/group). Compared to wild-type, we found that in TNFR1-KO, but not in TNFR2-KO mouse hearts, collagen deposition was attenuated, as was cardiac α-smooth muscle actin protein (a marker for activated fibroblasts). When we isolated viable cardiac fibroblasts and characterized them by flow cytometry, we found that Ang-II infusion in TNFR1-KO, but not in TNFR2-KO, resulted in a marked decrease of CD34 + /CD45 + cells. Quantitative RT-PCR demonstrated a striking reduction of type 1 and 3 collagen, as well of MCP-1 mRNA expression in TNFR1-KO mouse hearts. Further measurements of cardiovascular parameters indicated that TNFR1-KO animals developed lesser Ang-II-mediated LV remodeling, smaller changes in E-linear deceleration times/rates over time, and displayed a lower Tei index (a heart rate independent marker of cardiac function), indicating less stiffness in TNFR1-KO hearts compared to wild-type and TNFR2-KO hearts. The data suggest that Ang-II-dependent cardiac fibrosis requires TNF and its signaling through TNFR1 which enhances the induction of MCP-1 and uptake of monocytic fibroblast precursors that are associated with reactive fibrosis and cardiac remodeling and function.

scholarly journals The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1791
Author(s):  
Ana Bajc Česnik ◽  
Helena Motaln ◽  
Boris Rogelj
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Rna Binding ◽  
Neurodegenerative Disorder ◽  
Low Complexity ◽  
Wild Type ◽  
Disease Heterogeneity ◽  
Cytoplasmic Inclusions ◽  
The Impact ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

scholarly journals Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

2005 ◽  
Vol 25 (16) ◽  
pp. 7260-7269 ◽  
Author(s):  
Valerie Blanc ◽  
Jeffrey O. Henderson ◽  
Elizabeth P. Newberry ◽  
Susan Kennedy ◽  
Jianyang Luo ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cytidine Deaminase ◽  
Mrna Editing ◽  
Targeted Deletion ◽  
Apob Mrna ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Binding Subunit ◽  
Apparently Healthy

ABSTRACT apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf +/ − mice were apparently healthy and fertile, no viable acf − / − mice were identified. Mutant acf − / − embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf − / − blastocysts were detectable following implantation at E4.5, and isolated acf − / − blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

scholarly journals Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

2004 ◽  
Vol 24 (17) ◽  
pp. 7392-7401 ◽  
Author(s):  
Jan Medenbach ◽  
Silke Schreiner ◽  
Sunbin Liu ◽  
Reinhard Lührmann ◽  
Albrecht Bindereif
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Protein A ◽  
Tetratricopeptide Repeat ◽  
Terminal Sequence ◽  
Recognition Motifs ◽  
Tpr Domain

ABSTRACT After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

scholarly journals Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival

2009 ◽  
Vol 77 (7) ◽  
pp. 2783-2794 ◽  
Author(s):  
Scarlett Geunes-Boyer ◽  
Timothy N. Oliver ◽  
Guilhem Janbon ◽  
Jennifer K. Lodge ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Defense Mechanisms ◽  
Protein A ◽  
Surfactant Protein ◽  
Immune Defense ◽  
Surfactant Protein D ◽  
Wild Type ◽  
Phagocytic Cells

ABSTRACT Cryptococcus neoformans is a facultative intracellular opportunistic pathogen and the leading cause of fungal meningitis in humans. In the absence of a protective cellular immune response, the inhalation of C. neoformans cells or spores results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan, which constitutes approximately 90% of the capsular material. In the lungs, surfactant protein A (SP-A) and SP-D contribute to immune defense by facilitating the aggregation, uptake, and killing of many microorganisms by phagocytic cells. We hypothesized that SP-D plays a role in C. neoformans pathogenesis by binding to and enhancing the phagocytosis of the yeast. Here, the abilities of SP-D to bind to and facilitate the phagocytosis and survival of the wild-type encapsulated strain H99 and the cap59Δ mutant hypocapsular strain are assessed. SP-D binding to cap59Δ mutant cells was approximately sixfold greater than binding to wild-type cells. SP-D enhanced the phagocytosis of cap59Δ cells by approximately fourfold in vitro. To investigate SP-D binding in vivo, SP-D−/− mice were intranasally inoculated with Alexa Fluor 488-labeled cap59Δ or H99 cells. By confocal microscopy, a greater number of phagocytosed C. neoformans cells in wild-type mice than in SP-D−/− mice was observed, consistent with in vitro data. Interestingly, SP-D protected C. neoformans cells against macrophage-mediated defense mechanisms in vitro, as demonstrated by an analysis of fungal viability using a CFU assay. These findings provide evidence that C. neoformans subverts host defense mechanisms involving surfactant, establishing a novel virulence paradigm that may be targeted for therapy.

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