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Improved RNA modification mapping of cellular non-coding RNAs using C- and U-specific RNases.

Priti Thakur1, Mariana Estevez, Peter A Lobue

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New ribonucleases (RNases) cusativin and MC1 enhance RNA modification mapping by improving sequence coverage and distinguishing modifications. These tools aid in characterizing RNA sequences for structural and functional studies.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • RNA modification mapping is crucial for understanding RNA function.
  • Current methods using ribonucleases (RNases) like T1, U2, and A have limitations in sequence coverage and distinguishing certain modifications.
  • Accurate mapping requires digesting RNA into smaller fragments for analysis, often using liquid chromatography-mass spectrometry (LC-MS).

Purpose of the Study:

  • To introduce and evaluate novel nucleobase-specific ribonucleases, cusativin (cytosine-specific) and MC1 (uracil-specific), for RNA modification mapping.
  • To assess the ability of these new RNases to improve sequence coverage and confidence in mapping RNA modifications.
  • To demonstrate the utility of these enzymes in analyzing complex RNA samples like transfer RNA (tRNA) and ribosomal RNA (rRNA).

Main Methods:

  • Utilized E. coli tRNA and rRNA as model systems.
  • Employed complementary nucleobase-specific ribonucleases: cusativin (C-specific) and MC1 (U-specific) alongside traditional RNases (T1, U2, A).
  • Analyzed digestion products using liquid chromatography-mass spectrometry (LC-MS) to map RNA modifications.

Main Results:

  • Cusativin and MC1 generated unique digestion products, enhancing mapping in G-rich and pyrimidine-rich regions.
  • These enzymes facilitated differentiation between cytosine (C) and uracil (U) modifications and distinguished C to U sequence differences.
  • Combined with RNase T1, these new enzymes improved sequence coverage to over 75% for both tRNA and a large rRNA molecule (23S rRNA).
  • High confidence mapping of multiple modifications was achieved with minimal dependence on RNA abundance.

Conclusions:

  • The complementary ribonucleases cusativin and MC1 significantly improve RNA modification mapping efficiency and accuracy.
  • These enzymes increase sequence coverage and enable confident localization of modifications in challenging RNA regions.
  • Wider adoption of these tools can accelerate the characterization of modified RNAs, aiding the understanding of their roles in biological systems.