Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

ABSTRACT RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

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SUMO-1 Modification Alters ADAR1 Editing Activity

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0536 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5115-5126 ◽

Cited By ~ 75

Author(s):

Joana M.P. Desterro ◽

Liam P. Keegan ◽

Ellis Jaffray ◽

Ron T. Hay ◽

Mary A. O'Connell ◽

...

Keyword(s):

Rna Editing ◽

Nucleolar Localization ◽

Dense Fibrillar Component ◽

Wild Type ◽

Fibrillar Center ◽

Editing Activity ◽

Fibrillar Component ◽

Editing Enzyme

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.

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Patterns of RNA Editing in Newcastle Disease Virus Infections

Viruses ◽

10.3390/v12111249 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1249

Author(s):

Archana Jadhav ◽

Lele Zhao ◽

Alice Ledda ◽

Weiwei Liu ◽

Chan Ding ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Rna Editing ◽

Disease Virus ◽

Newcastle Disease ◽

Structural Proteins ◽

Virus Infections ◽

Mrna Editing ◽

P Protein

The expression of accessory non-structural proteins V and W in Newcastle disease virus (NDV) infections depends on RNA editing. These proteins are derived from frameshifts of the sequence coding for the P protein via co-transcriptional insertion of one or two guanines in the mRNA. However, a larger number of guanines can be inserted with lower frequencies. We analysed data from deep RNA sequencing of samples from in vitro and in vivo NDV infections to uncover the patterns of mRNA editing in NDV. The distribution of insertions is well described by a simple Markov model of polymerase stuttering, providing strong quantitative confirmation of the molecular process hypothesised by Kolakofsky and collaborators three decades ago. Our results suggest that the probability that the NDV polymerase would stutter is about 0.45 initially, and 0.3 for further subsequent insertions. The latter probability is approximately independent of the number of previous insertions, the host cell, and viral strain. However, in LaSota infections, we also observe deviations from the predicted V/W ratio of about 3:1 according to this model, which could be attributed to deviations from this stuttering model or to further mechanisms downregulating the abundance of W protein.

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ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1904 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1904-C1916 ◽

Cited By ~ 38

Author(s):

Shrikant Anant ◽

Debnath Mukhopadhyay ◽

Vakadappu Sankaranand ◽

Susan Kennedy ◽

Jeffrey O. Henderson ◽

...

Keyword(s):

Rna Editing ◽

Rna Binding ◽

Cytidine Deaminase ◽

Binding Activity ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Mrna Editing ◽

Apob Mrna

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.

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RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Molecular and Cellular Biology ◽

10.1128/mcb.01374-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 122-130 ◽

Cited By ~ 70

Author(s):

Jason Carnes ◽

James Raffaello Trotter ◽

Adam Peltan ◽

Michele Fleck ◽

Kenneth Stuart

Keyword(s):

Trypanosoma Brucei ◽

Rna Editing ◽

Editing Site ◽

Unexpected Finding ◽

Rnase Iii ◽

Growth And Survival ◽

Insertion And Deletion ◽

Additional Level

ABSTRACT Trypanosoma brucei has three distinct ∼20S editosomes that catalyze RNA editing by the insertion and deletion of uridylates. Editosomes with the KREN1 or KREN2 RNase III type endonucleases specifically cleave deletion and insertion editing site substrates, respectively. We report here that editosomes with KREPB2, which also has an RNase III motif, specifically cleave cytochrome oxidase II (COII) pre-mRNA insertion editing site substrates in vitro. Conditional repression and mutation studies also show that KREPB2 is an editing endonuclease specifically required for COII mRNA editing in vivo. Furthermore, KREPB2 expression is essential for the growth and survival of bloodstream forms. Thus, editing in T. brucei requires at least three compositionally and functionally distinct ∼20S editosomes, two of which distinguish between different insertion editing sites. This unexpected finding reveals an additional level of complexity in the RNA editing process and suggests a mechanism for how the selection of sites for editing in vivo is controlled.

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Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+Signaling

BioMed Research International ◽

10.1155/2017/3691349 ◽

2017 ◽

Vol 2017 ◽

pp. 1-16 ◽

Cited By ~ 6

Author(s):

Nahed El-Najjar ◽

Rashmi P. Kulkarni ◽

Nancy Nader ◽

Rawad Hodeify ◽

Khaled Machaca

Keyword(s):

Insulin Resistance ◽

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Vascular Smooth Muscle ◽

Complex Disease ◽

Prolonged Exposure ◽

In Vitro System ◽

A7r5 Cells

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+signaling, including most prominently an inhibition of the passive ER Ca2+leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+signaling machinery are different.

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Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

Eukaryotic Cell ◽

10.1128/ec.00284-06 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2214-2221 ◽

Cited By ~ 53

Author(s):

Lois M. Douglas ◽

Li Li ◽

Yang Yang ◽

A. M. Dranginis

Keyword(s):

Saccharomyces Cerevisiae ◽

Biofilm Formation ◽

In Vitro System ◽

Glycosylphosphatidylinositol Anchor ◽

Fungal Adhesion ◽

Very High ◽

Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

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Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4746 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4746-4749 ◽

Cited By ~ 97

Author(s):

D I Chasman ◽

J Leatherwood ◽

M Carey ◽

M Ptashne ◽

R D Kornberg

Keyword(s):

Rna Polymerase Ii ◽

Fusion Proteins ◽

In Vitro System ◽

Transcription System ◽

Natural Mechanism ◽

Transcription In Vitro ◽

Rna Polymerase Ii Transcription ◽

Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

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Accurate transcription of a plant mitochondrial gene in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2035-2039.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2035-2039

Author(s):

P J Hanic-Joyce ◽

M W Gray

Keyword(s):

Mitochondrial Gene ◽

Plant Mitochondria ◽

In Vitro Transcription ◽

Dna Templates ◽

In Vitro System ◽

Transcription System ◽

Translation Initiation Codon ◽

Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

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Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽

10.1017/s0043174500055417 ◽

1980 ◽

Vol 28 (3) ◽

pp. 334-340 ◽

Cited By ~ 26

Author(s):

Luanne M. Deal ◽

J. T. Reeves ◽

B. A. Larkins ◽

F. D. Hess

Keyword(s):

Protein Synthesis ◽

Sand Culture ◽

Leucine Incorporation ◽

Insoluble Protein ◽

In Vitro System ◽

A Cell ◽

Protein Incorporation ◽

Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

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