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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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Intact DNA strands can be found in fossils, while scientists sometimes struggle to keep RNA intact under laboratory conditions. The structural variations between RNA and DNA underlie the differences in their stability and longevity. Because DNA is double-stranded, it is inherently more stable. The single-stranded structure of RNA is less stable but also more flexible and can form weak internal bonds. Additionally, most RNAs in the cell are relatively short, while DNA can be up to 250 million...
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The histone proteins in the nucleosomes are post-translationally modified (PTM) to increase or decrease access to DNA. The commonly observed PTMs are methylation, acetylation, phosphorylation, and ubiquitination of lysine amino acids in the histone H3 tail region. These histone modifications have specific meaning for the cell. Hence, they are called "histone code". The protein complex involved in histone modification is termed as "reader-writer" complex.
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The histone proteins have a flexible N-terminal tail extending out from the nucleosome. These histone tails are often subjected to post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Particular combinations of these modifications form “histone codes” that influence the chromatin folding and tissue-specific gene expression.
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Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen
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Improving RNA modification mapping sequence coverage by LC-MS through a nonspecific RNase U2-E49A mutant.

Beulah Solivio1, Ningxi Yu1, Balasubrahmanyam Addepalli1

  • 1Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, United States.

Analytica Chimica Acta
|September 27, 2018
PubMed
Summary

Researchers developed a mutant ribonuclease U2 (RNase U2) enzyme that randomly cleaves RNA. This engineered enzyme improves RNA modification mapping accuracy and efficiency using mass spectrometry.

Keywords:
Nonspecific endoribonucleasePost-transcriptional modificationsRibonuclease U2tRNA sequencing

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Analytical Chemistry

Background:

  • Ribonucleases (RNases) are crucial for RNA analysis, but their high specificity can limit comprehensive mapping of RNA modifications.
  • Current methods for RNA modification mapping often require multiple enzymatic digestions and may not achieve full sequence coverage.

Purpose of the Study:

  • To identify and characterize a mutant RNase U2 with altered specificity for improved RNA modification mapping.
  • To demonstrate the utility of the engineered RNase U2 mutant in achieving high-sequence coverage for modified RNA analysis.

Main Methods:

  • Site-saturation mutagenesis was employed to generate RNase U2 mutants.
  • Oligonucleotide substrates were used to test enzyme activity and specificity.
  • Liquid chromatography tandem mass spectrometry (LC-MS/MS) was utilized for RNA modification mapping.

Main Results:

  • An RNase U2 E49A mutant exhibiting limited specificity and RNA undercutting was identified.
  • Optimized digestion conditions with the E49A mutant yielded long, overlapping RNA fragments.
  • 100% sequence coverage of modified RNAs was achieved using a single digestion with the mutant enzyme.

Conclusions:

  • The RNase U2 E49A mutant enables more accurate and efficient RNA modification mapping compared to traditional base-specific RNases.
  • This engineered enzyme significantly enhances the analytical capabilities for studying RNA modifications.