Comparative Activity of Adenosine Deaminase Acting on RNA (ADARs) Isoforms for Correction of Genetic Code in Gene Therapy

Introduction: Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD. Materials and Methods: We measured the binding affinity of the artificial enzyme system on a Biacore ™ X100. ADARs usually target dsRNA, so we designed a guide RNA complementary to the target RNA, and then fused the guide sequence to the MS2 stem-loop. A mutated amber (TAG) stop codon at 58 amino acid (TGG) of EGFP was targeted. After transfection of these three factors into HEK 293 cells, we observed fluorescence signals of various intensities. Results: ADAR2-long without the Alu-cassette yielded a much higher fluorescence signal than ADAR2-long with the Alu-cassette. With another isoform, ADAR2-short, which is 81 bp shorter at the C-terminus, the fluorescence signal was undetectable. A single amino acid substitution of ADAR2-long-DD (E488Q) rendered the enzyme more active than the wild type. The results of fluorescence microscopy suggested that ADAR1-DD is more active than ADAR2-long-DD. Western blots and sequencing confirmed that ADAR1-DD was more active than any other DD. Conclusion: This study provides information that should facilitate the rational use of ADAR variants for genetic restoration and treatment of genetic diseases.

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The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy

Genome ◽

10.1139/gen-2021-0010 ◽

2021 ◽

Author(s):

Sakura Hayashi ◽

Takuji Tsukiyama ◽

Atsuo Iida ◽

Masato Kinoshita ◽

Akihiko Koga

Keyword(s):

Amino Acid ◽

Stop Codon ◽

Early Stage ◽

Single Amino Acid ◽

Base Substitution ◽

Unique Element ◽

Medaka Fish ◽

Medaka Genome ◽

Single Amino Acid Change ◽

Local Sequence

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained 3 local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that the transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost whole decay process that progresses in natural populations.

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Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.358 ◽

1995 ◽

Vol 15 (1) ◽

pp. 358-364 ◽

Cited By ~ 61

Author(s):

S R Green ◽

L Manche ◽

M B Mathews

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Rna Binding ◽

Rna Binding Proteins ◽

Alpha Helix ◽

Binding Domain ◽

Single Amino Acid ◽

Double Stranded Rna ◽

C Terminus ◽

Rna Binding Domain

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

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Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase

Biochemical Journal ◽

10.1042/bj3050439 ◽

1995 ◽

Vol 305 (2) ◽

pp. 439-444 ◽

Cited By ~ 7

Author(s):

T M Johnson ◽

H P Kocher ◽

R C Anderson ◽

G M Nemecek

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Heterologous Expression ◽

Stop Codon ◽

Expression System ◽

Breast Muscle ◽

Cdna Clones ◽

Carnitine Acetyltransferase ◽

Western Blots ◽

Pigeon Liver

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5′ and 3′ ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3′-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.

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Two Trypanosome-Specific Proteins Are Essential Factors for 5S rRNA Abundance and Ribosomal Assembly in Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00119-07 ◽

2007 ◽

Vol 6 (10) ◽

pp. 1766-1772 ◽

Cited By ~ 19

Author(s):

Kristina M. Hellman ◽

Martin Ciganda ◽

Silvia V. Brown ◽

Jinlei Li ◽

William Ruyechan ◽

...

Keyword(s):

Amino Acid ◽

Trypanosoma Brucei ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Rna Binding Proteins ◽

5S Rrna ◽

Single Amino Acid ◽

Ribosomal Subunits ◽

Morphological Alterations ◽

Specific Proteins

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.

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Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human ‘knockout’

Acta Endocrinologica ◽

10.1530/eje-13-0094 ◽

2013 ◽

Vol 169 (1) ◽

pp. K1-K7 ◽

Cited By ~ 5

Author(s):

D T Ward ◽

M Z Mughal ◽

M Ranieri ◽

M M Dvorak-Ewell ◽

G Valenti ◽

...

Keyword(s):

Parathyroid Gland ◽

Transmembrane Domain ◽

Stop Codon ◽

Single Amino Acid ◽

Total Parathyroidectomy ◽

Calcium Sensing Receptor ◽

Hek 293 ◽

Hek 293 Cells ◽

Calcium Sensing ◽

293 Cells

ObjectiveLoss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaRR392X), in effect a full CAR ‘knockout’.Case reportThe infant (daughter of distant cousins) presented with hypercalcaemia (5.5–6 mmol/l corrected calcium (2.15–2.65)) and elevated PTH concentrations (650–950 pmol/l (12–81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels.Design and methodsThe R392X stop codon was inserted into human CAR and the resulting mutant (CaRR392X) expressed transiently in HEK-293 cells.ResultsCaRR392X expressed as a 54 kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaRR392X-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity.ConclusionsIntriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10–71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

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Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution

Nature Structural & Molecular Biology ◽

10.1038/s41594-020-0436-2 ◽

2020 ◽

Vol 27 (7) ◽

pp. 678-682 ◽

Cited By ~ 3

Author(s):

Jong Woo Bae ◽

S. Chul Kwon ◽

Yongwoo Na ◽

V. Narry Kim ◽

Jong-Seo Kim

Keyword(s):

Amino Acid ◽

Binding Site ◽

Rna Binding ◽

Single Amino Acid ◽

Site Mapping

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Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.762-767.1985 ◽

1985 ◽

Vol 5 (4) ◽

pp. 762-767

Author(s):

S H Orkin ◽

S C Goff ◽

W N Kelley ◽

P E Daddona

Keyword(s):

Amino Acid ◽

Adenosine Deaminase ◽

Cultured Cells ◽

Simian Virus 40 ◽

Lymphoid Cells ◽

Single Amino Acid ◽

Molecular Defect ◽

Unexpected Finding ◽

Naturally Occurring ◽

Ada Protein

Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.

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Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software

10.1101/2021.06.16.448102 ◽

2021 ◽

Author(s):

Xiaoping Yang ◽

Andrew L Hong ◽

Ted Sharpe ◽

Andrew O Giacomelli ◽

Robert E Lintner ◽

...

Keyword(s):

Amino Acid ◽

Stop Codon ◽

Direct Sequencing ◽

Amino Acid Position ◽

Single Amino Acid ◽

Library Design ◽

Protein Variant ◽

Reading Frame ◽

Detection Software ◽

Small Orfs

Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously. A variant library targeting a full-length ORF typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a single codon variation separates a variant from the template ORF, variant quantification presents the most profound challenge. Efforts such as dividing a library into sub-libraries for direct sequencing or using a tag-directed subassembly approach are practical only with small ORFs. Our approach, on the other hand, features single-pool libraries for all genes up to 3600bp (EGFR), and an enhanced variant-detecting toolkit. Having succeeded in processing screens of ~20 ORF variant libraries, this tool calls variants reliably, and also presents variant annotations in datafiles to enable analyses that have in turn reshaped our strategies governing library design, screen deconvolution, sequencing and its analysis.

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A Single Amino Acid Change in Turnip Crinkle Virus Movement Protein p8 Affects RNA Binding and Virulence onArabidopsis thaliana

Journal of Virology ◽

10.1128/jvi.72.7.6247-6250.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 6247-6250 ◽

Cited By ~ 27

Author(s):

Kristin K. Wobbe ◽

Muslum Akgoz ◽

D’Maris Amick Dempsey ◽

Daniel F. Klessig

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Rna Binding ◽

Movement Protein ◽

Single Amino Acid ◽

Turnip Crinkle Virus ◽

In Planta ◽

Viral Movement Protein ◽

Single Amino Acid Change ◽

Viral Movement

ABSTRACT Comparison of the symptoms caused by turnip crinkle virus strain M (TCV-M) and TCV-B infection of a resistant Arabidopsis thaliana line termed Di-17 demonstrates that TCV-B has a greater ability to spread in planta. This ability is due to a single amino acid change in the viral movement protein p8 and inversely correlates with p8 RNA binding affinity.

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A Peptide from Autoantigen La Blocks Poliovirus and Hepatitis C Virus Cap-Independent Translation and Reveals a Single Tyrosine Critical for La RNA Binding and Translation Stimulation

Journal of Virology ◽

10.1128/jvi.78.7.3763-3776.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3763-3776 ◽

Cited By ~ 34

Author(s):

Raquel E. Izumi ◽

Saumitra Das ◽

Bhaswati Barat ◽

Santanu Raychaudhuri ◽

Asim Dasgupta

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Lupus Erythematosus ◽

Rna Binding ◽

Single Amino Acid ◽

Conserved Sequence ◽

La Protein ◽

Hcv Ires

ABSTRACT La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal “La motif” which is required for efficient interaction of La with IRNA and viral 5′ untranslated region (5′-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5′-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5′-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.

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