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The poly(A) tail is a dynamic structure at the 3′- end of the majority of RNA polymerase II transcripts. It is a critical feature, particularly for mRNAs, as the length of the poly(A) tail regulates their translational efficiency and lifespan. The shortening of the tail is catalyzed by deadenylases that trim and finally remove it, triggering mRNA degradation. Conventional assays to evaluate and measure deadenylase activity rely on radiolabeling of the substrate while colorimetric alternatives lack sensitivity. In this chapter, we describe a poly(A)-shortening assay using a 5′-Cy3-labeled RNA substrate bearing a 3′-oligo(A) sequence. The reaction products representing substrates with shortened tails are analyzed by denaturing acrylamide electrophoresis and are visualized using a gel documentation system. This assay is efficient, it requires standard lab equipment, including a fluorescence-detecting gel documentation system, and presents a quantifiable and safer alternative to radioactivity-based methods for evaluating deadenylase activity.

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Assessing Deadenylation Activity by Polyacrylamide Gel Electrophoresis Using a Fluorescent RNA Substrate

  • 实验方案

  • 发表日期：

    2025年

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  • Rafailia A. A. Beta,
  • Nikolaos A. A. Balatsos

摘要

The poly(A) tail is a dynamic structure at the 3′- end of the majority of RNA polymerase II transcripts. It is a critical feature, particularly for mRNAs, as the length of the poly(A) tail regulates their translational efficiency and lifespan. The shortening of the tail is catalyzed by deadenylases that trim and finally remove it, triggering mRNA degradation. Conventional assays to evaluate and measure deadenylase activity rely on radiolabeling of the substrate while colorimetric alternatives lack sensitivity. In this chapter, we describe a poly(A)-shortening assay using a 5′-Cy3-labeled RNA substrate bearing a 3′-oligo(A) sequence. The reaction products representing substrates with shortened tails are analyzed by denaturing acrylamide electrophoresis and are visualized using a gel documentation system. This assay is efficient, it requires standard lab equipment, including a fluorescence-detecting gel documentation system, and presents a quantifiable and safer alternative to radioactivity-based methods for evaluating deadenylase activity.