Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.
Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA
RNA. 2024 Sep 16;30(10):1315-1327. doi: 10.1261/rna.080156.124.
Members of the 3'-5' RNA polymerase family, comprised of tRNA guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.
3′-5′ RNA 聚合酶家族的成员包括 tRNA 鸟苷酰转移酶（Thg1）和 Thg1 样蛋白（TLPs），它们在模板的指导下，催化 RNA 的反向合成，其方向与所有已知的 5′-3′ RNA 和 DNA 聚合酶相反。能够进行这种反应的酶的发现，使得人们有可能利用 3′-5′聚合酶在没有连接的情况下，通过模板聚合酶的添加，将核苷酸在后转录过程中掺入到核酸的 5′-端。迄今为止，这些酶的研究主要集中在高度结构化 RNA（如 tRNA 和其他非编码 RNA）的核苷酸添加上。因此，尚未阐明可能使 3′-5′聚合酶得到更广泛应用的 RNA 底物识别和核苷酸偏好的一般原则。在这里，我们研究了使用 Thg1 或 TLPs 将多个核苷酸掺入到短双链 RNA 底物的 5′-端的可行性，使用提供的模板 RNA 寡核苷酸来指导特定序列的 5′-端添加。使用优化的测定条件，我们证明了某些 TLPs 具有显著的能力，可以高度亲和地容纳具有不同长度的短 RNA 底物-模板双链体，从而能够以模板依赖性方式将多达八个碱基的所需核苷酸序列掺入到模型 RNA 底物的 5′-端。这项工作进一步推进了我们的目标，即将这个非典型的酶家族开发成一种通用的核酸 5′-端标记工具。
