Department of Biology, University of Nevada, Reno.
Department of Biology, University of Nevada, Reno;
J Vis Exp. 2024 Jan 12(203). doi: 10.3791/66116.
Polyadenylation is a crucial posttranscriptional modification that adds poly(A) tails to the 3' end of mRNA molecules. The length of the poly(A) tail is tightly regulated by cellular processes. Dysregulation of mRNA polyadenylation has been associated with abnormal gene expression and various diseases, including cancer, neurological disorders, and developmental abnormalities. Therefore, comprehending the dynamics of polyadenylation is vital for unraveling the complexities of mRNA processing and posttranscriptional gene regulation. This paper presents a method for measuring poly(A) tail lengths in RNA samples isolated from Drosophila larval brains and Drosophila Schneider S2 cells. We employed the guanosine/inosine (G/I) tailing approach, which involves the enzymatic addition of G/I residues at the 3' end of mRNA using yeast poly(A) polymerase. This modification protects the RNA's 3' end from enzymatic degradation. The protected full-length poly(A) tails are then reverse-transcribed using a universal antisense primer. Subsequently, PCR amplification is performed using a gene-specific oligo that targets the gene of interest, along with a universal sequence oligo used for reverse transcription. This generates PCR products encompassing the poly(A) tails of the gene of interest. Since polyadenylation is not a uniform modification and results in tails of varying lengths, the PCR products display a range of sizes, leading to a smear pattern on agarose gel. Finally, the PCR products are subjected to high-resolution capillary gel electrophoresis, followed by quantification using the sizes of the poly(A) PCR products and the gene-specific PCR product. This technique offers a straightforward and reliable tool for analyzing poly(A) tail lengths, enabling us to gain deeper insights into the intricate mechanisms governing mRNA regulation.
多聚腺苷酸化是一种至关重要的转录后修饰过程,它在 mRNA 分子的 3' 端添加多聚(A)尾。多聚(A)尾的长度受到细胞过程的严格调控。mRNA 多聚腺苷酸化的失调与异常基因表达和各种疾病有关,包括癌症、神经紊乱和发育异常。因此,理解多聚腺苷酸化的动态对于揭示 mRNA 处理和转录后基因调控的复杂性至关重要。本文介绍了一种从果蝇幼虫大脑和果蝇 Schneider S2 细胞分离的 RNA 样本中测量多聚(A)尾长度的方法。我们采用了鸟苷/肌苷(G/I)加尾方法,该方法涉及使用酵母多聚(A)聚合酶在 mRNA 的 3' 端添加 G/I 残基。这种修饰保护 RNA 的 3' 端免受酶的降解。然后,使用通用反义引物对受保护的全长多聚(A)尾进行逆转录。随后,使用针对感兴趣基因的基因特异性寡核苷酸和用于逆转录的通用序列寡核苷酸进行 PCR 扩增。这产生了包含感兴趣基因的多聚(A)尾的 PCR 产物。由于多聚腺苷酸化不是一种均匀的修饰,并且会产生长度不同的尾巴,因此 PCR 产物显示出一系列大小,在琼脂糖凝胶上呈现出弥散模式。最后,将 PCR 产物进行高分辨率毛细管凝胶电泳,然后使用多聚(A)PCR 产物和基因特异性 PCR 产物的大小进行定量。该技术提供了一种简单可靠的分析多聚(A)尾长度的工具,使我们能够更深入地了解调节 mRNA 的复杂机制。