scholarly journals A streamlined protocol for the detection of mRNA–sRNA interactions using AMT-crosslinking in vitro

BioTechniques ◽  
2019 ◽  
Vol 67 (4) ◽  
pp. 178-183
Author(s):  
Rebecca Kirsch ◽  
V Janett Olzog ◽  
Sonja Bonin ◽  
Christina E Weinberg ◽  
Heike Betat ◽  
...  
Keyword(s):  
Local Concentration ◽  
Crosslinking Reaction ◽  
Interacting Proteins ◽  
Rna Ligase ◽  
Proximity Ligation ◽  
T4 Rna Ligase ◽  
Interaction Partners ◽  
Cyclic Phosphate ◽  
Direct Fusion

Until recently, RNA–RNA interactions were mainly identified by crosslinking RNAs with interacting proteins, RNA proximity ligation and deep sequencing. Recently, AMT-based direct RNA crosslinking was established. Yet, several steps of these procedures are rather inefficient, reducing the output of identified interaction partners. To increase the local concentration of RNA ends, interacting RNAs are often fragmented. However, the resulting 2′,3′-cyclic phosphate and 5′-OH ends are not accepted by T4 RNA ligase and have to be converted to 3′-OH and 5′-phosphate ends. Using an artificial mRNA/sRNA pair, we optimized the workflow downstream of the crosslinking reaction in vitro. The use of a tRNA ligase allows direct fusion of 2′,3′-cyclic phosphate and 5′-OH RNA ends.

The characterization and translation in vitro of rabbit globin messenger RNA and messenger ribonucleoprotein labelled with bacteriophage-T4 RNA ligase and 5′-[32P]phosphocytidine 3′-phosphate

1982 ◽  
Vol 10 (2) ◽  
pp. 91-92
Author(s):  
N. SHAUN B. THOMAS ◽  
PHILIP D. BUTCHER ◽  
JOHN M. KELLY ◽  
ROBERT A. COX ◽  
HENRY R. V. ARNSTEIN
Keyword(s):  
Messenger Rna ◽  
Bacteriophage T4 ◽  
Rna Ligase ◽  
Messenger Ribonucleoprotein ◽  
T4 Rna Ligase

Synthesis of aspartic acid derivatives useful for the preparation of misacylated transfer RNAs

2000 ◽  
Vol 78 (6) ◽  
pp. 884-891 ◽  
Author(s):  
Michiel Lodder ◽  
Curtis F Crasto ◽  
Andrei L Laikhter ◽  
Haoyun An ◽  
Tuncer Arslan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Side Chain ◽  
Transfer Rnas ◽  
Rna Ligase ◽  
T4 Rna Ligase ◽  
In Vitro Transcripts ◽  
Derivatives Of

Several derivatives of aspartic acid were protected on Nα as their NVOC derivatives, and on the side chain carboxylates as nitroveratryl esters. Following activation as the cyanomethyl esters, these fully protected aspartate derivatives were converted to the respective pdCpA esters. The protected aspartyl-pdCpA esters were then utilized as substrates for T4 RNA ligase in the presence of in vitro transcripts of tRNA lacking the pCpA dinucleotide normally found at the 3'-end. In this fashion, several misacylated tRNAs were prepared; following photolytic deprotection, these were employed successfully for incorporation into proteins at predetermined positions.Key words: aminoacylated nucleotides, amino acid protection, protein synthesis, tRNA activation.

scholarly journals In vitro selection of optimal DNA substrates for T4 RNA ligase.

1993 ◽  
Vol 90 (4) ◽  
pp. 1576-1579 ◽  
Author(s):  
K. Harada ◽  
L. E. Orgel
Keyword(s):  
In Vitro Selection ◽  
Rna Ligase ◽  
T4 Rna Ligase ◽  
Dna Substrates ◽  
Selection Of

Regulation of RNA helicase activity: principles and examples

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Pascal Donsbach ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Wide Spectrum ◽  
Mrna Translation ◽  
Rna Degradation ◽  
Rna Helicases ◽  
Interaction Partners ◽  
Rna Duplexes ◽  
Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

Preparation of '2,('3)-O-Acyl-pCpA derivatives as substrates for T4 RNA ligase-mediated “chemical aminoacylation”

Tetrahedron ◽  
1984 ◽  
Vol 40 (1) ◽  
pp. 87-94 ◽  
Author(s):  
T.G. Heckler ◽  
L.H. Chang ◽  
Y. Zama ◽  
T. Naka ◽  
S.M. Hecht
Keyword(s):  
Rna Ligase ◽  
T4 Rna Ligase

Detection of single-base mutation in RNA using T4 RNA ligase-based nick-joining or DNAzyme-based nick-generation

2011 ◽  
Vol 414 (2) ◽  
pp. 303-305 ◽  
Author(s):  
Kkothanahreum Park ◽  
Bo-Ra Choi ◽  
Yea Seul Kim ◽  
Seonmi Shin ◽  
Sang Soo Hah ◽  
...  
Keyword(s):  
Rna Ligase ◽  
Single Base ◽  
T4 Rna Ligase

scholarly journals AHR:IKAROS Interaction Promotes Platelet Biogenesis in Response to SR1

Reports ◽  
2021 ◽  
Vol 4 (1) ◽  
pp. 7
Author(s):  
Lea Mallo ◽  
Valentin Do Sacramento ◽  
Christian Gachet ◽  
Susan Chan ◽  
Philippe Kastner ◽  
...  
Keyword(s):  
High Capacity ◽  
Proximity Ligation Assay ◽  
Physical Interaction ◽  
Short Report ◽  
Production Engineering ◽  
Platelet Production ◽  
Proximity Ligation ◽  
Aryl Hydrocarbon ◽  
Dependent Pathway

In vitro, the differentiation of megakaryocytes (MKs) is improved by aryl-hydrocarbon receptor (AHR) antagonists such as StemRegenin 1 (SR1), an effect physiologically recapitulated by the presence of stromal mesenchymal cells (MSC). This inhibition promotes the amplification of a CD34+CD41low population able to mature as MKs with a high capacity for platelet production. In this short report, we showed that the emergence of the thrombocytogenic precursors and the enhancement of platelet production triggered by SR1 involved IKAROS. The downregulation/inhibition of IKAROS (shRNA or lenalidomide) significantly reduced the emergence of SR1-induced thrombocytogenic population, suggesting a crosstalk between AHR and IKAROS. Interestingly, using a proximity ligation assay, we could demonstrate a physical interaction between AHR and IKAROS. This interaction was also observed in the megakaryocytic cells differentiated in the presence of MSCs. In conclusion, our study revealed a previously unknown AHR/ IKAROS -dependent pathway which prompted the expansion of the thrombocytogenic precursors. This AHR- IKAROS dependent checkpoint controlling MK maturation opens new perspectives to platelet production engineering.

scholarly journals Dual Mechanisms whereby a Broken RNA End Assists the Catalysis of Its Repair by T4 RNA Ligase 2

2005 ◽  
Vol 280 (25) ◽  
pp. 23484-23489 ◽  
Author(s):  
Jayakrishnan Nandakumar ◽  
Stewart Shuman
Keyword(s):  
Rna Ligase ◽  
T4 Rna Ligase

scholarly journals SGTA associates with intracellular aggregates in neurodegenerative diseases.

2020 ◽  
Author(s):  
Shun Kubota ◽  
Hiroshi Doi ◽  
Shigeru Koyano ◽  
Kenichi Tanaka ◽  
Shingo Ikeda ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Molecular Mechanisms ◽  
In Vitro Study ◽  
Interacting Proteins ◽  
Interacting Protein ◽  
Cytoplasmic Inclusions ◽  
Specific Proteins ◽  
Intracellular Aggregates ◽  
Polyq Diseases

Abstract Intracellular aggregates are a common pathological hallmark of neurodegenerative diseases such as polyglutamine (polyQ) diseases, amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and multiple system atrophy (MSA). Aggregates are mainly formed by aberrant disease-specific proteins and are accompanied by accumulation of other aggregate-interacting proteins. Although aggregate-interacting proteins have been considered to modulate the formation of aggregates and to be involved in molecular mechanisms of disease progression, the components of aggregate-interacting proteins remain unknown. In this study, we showed that small glutamine-rich tetratricopeptide repeat-containing protein alfa (SGTA) is an aggregate-interacting protein in neurodegenerative diseases. Immunohistochemistry showed that SGTA interacted with intracellular aggregates in Huntington disease (HD) cell models and neurons of HD model mice. We also revealed that SGTA colocalized with intracellular aggregates in postmortem brains of patients with polyQ diseases including spinocerebellar ataxia (SCA)1, SCA2, SCA3, and dentatorubral–pallidoluysian atrophy. In addition, SGTA colocalized with glial cytoplasmic inclusions in the brains of MSA patients, whereas no accumulation of SGTA was observed in neurons of PD and ALS patients. In vitro study showed that SGTA bound to polyQ aggregates through its C-terminal domain and SGTA overexpression reduced intracellular aggregates. These results suggest that SGTA may play a role in the formation of aggregates and may act as potential modifier of molecular pathological mechanisms of polyQ diseases and MSA.

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