Shenzhen Key Laboratory of Microbial Genetic Engineering, Vascular Disease Research Center, College of Life Sciences and Oceanography, Guangdong Provincial Key Laboratory of Regional Immunity and Disease, Shenzhen University, 1066 Xueyuan Street, Nanshan District, Shenzhen 518055, Guangdong, China.
The School of Medicine, Life and Health Sciences (MED | LHS), Chinese University of Hong Kong, Shenzhen, No. 2001 Longxiang Blvd., Longgang District, Shenzhen 518172, Guangdong, China.
Brief Bioinform. 2023 Sep 22;24(6). doi: 10.1093/bib/bbad374.
Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.
动态调节多聚(A)尾是控制真核 mRNA 翻译和稳定性的关键机制。要全面了解这种调控是如何发生的,需要使用高通量测序平台对多聚(A)尾转录本进行无偏的丰度定量,并对简单的多聚(A)长度进行测量。目前的方法由于设置复杂和详细的文库制备计划而存在局限性。为了解决这个问题,我们引入了基于中心极限定理(CLT)的 RNA 测序(CLT-seq),这是一种简单直接的同源聚核苷酸测序方法。在 CLT-seq 中,无锚定 oligo(dT)引物快速结合和从 poly(A)尾字符串的任何位置解离,导致以相等的概率进行位置定向逆转录。CLT 机制使合成的 poly(T)长度,对应于 poly(A)尾的模板片段,呈正态分布。基于拟合良好的伪高斯衍生的 poly(A)-poly(T)转换模型,通过矩阵运算从获得的 poly(T)-长度分布中重建实际的 poly(A)-尾轮廓。CLT-seq 遵循一个简单的程序,不需要与 RNA 相关的预处理、富集或选择,并且 CLT 缩短的 poly(T)延伸与现有的测序平台更兼容。这种概念验证方法促进了直接的同源聚核苷酸碱基调用,并具有无偏倚的 RNA-seq 特征。因此,CLT-seq 提供了无偏、稳健和经济高效的转录组范围的 poly(A)-尾分析。我们证明,在最常见的 Illumina 平台上进行 CLT-seq,可以在人类细胞环境中以转录组范围的规模可靠地进行 poly(A)-尾分析。我们发现,poly(A)-尾调节的 ncRNA 调节经历了一个与 mRNA 调节相似的动态、复杂的过程。总的来说,CLT-seq 提供了一种简化、有效和经济的方法来研究 poly(A)-尾调节,这可能对理解基因表达和识别治疗靶点具有重要意义。