Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Nucleic Acids Res. 2019 Sep 19;47(16):8755-8769. doi: 10.1093/nar/gkz576.
Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3'-5' exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R can fail to digest >20% of all highly expressed linear RNAs, but these shortcomings can largely be overcome. RNAs with highly structured 3' ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many polyadenylated mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases.
数以千计的真核生物蛋白编码基因产生具有共价连接末端的环状 RNA，并且对核酸外切酶的降解具有抗性。为了证明它们的环状结构，并在生化上富集这些转录本，在该领域中，使用 3'-5'核酸外切酶 RNase R 已经成为标准方法。在这里，我们证明涉及 RNase R 的标准方案可能无法消化超过 20%的所有高表达线性 RNA，但这些缺点在很大程度上可以克服。具有高度结构化 3'末端的 RNA，包括 snRNA 和组蛋白 mRNA，天然对 RNase R 具有抗性，但一旦在其末端添加聚（A）尾巴，就可以有效地降解。此外，RNase R 在许多多聚腺苷酸化 mRNA 的主体中停滞不前，尤其是在先前注释为 G-四链体 (G4) 结构的富含 G 序列处。在用反应缓冲液中的 Li+替代 K+（稳定 G4s）后，我们发现 RNase R 现在能够穿过这些序列并完全降解整个 mRNA。总的来说，我们的结果为当前用于分离环状 RNA 的方法提供了重要的改进，以及一种揭示可能自然抑制细胞核酸外切酶降解的 RNA 结构的方法。
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