Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen B. burgdorferi [3′RNA-seq]. Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen B. burgdorferi [3′RNA-seq]

Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. We employed complementary RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi – the etiological agent of Lyme disease. By systematically mapping B. burgdorferi RNA ends at single nucleotide resolution, we delineated complex gene arrangements and operons and mapped untranslated regions (UTRs) and small RNAs (sRNAs). We experimentally tested modes of B. burgdorferi transcription termination and compared our findings to observations in E. coli, P. aeruginosa, and B. subtilis. We discovered 63% of B. burgdorferi RNA 3′ ends map upstream or internal to open reading frames (ORFs), suggesting novel mechanisms of regulation. Northern analysis confirmed the presence of stable 5′ derived RNAs from mRNAs encoding gene products involved in the unique infectious cycle of B. burgdorferi. We suggest these RNAs resulted from premature termination and regulatory events, including forms of cis-acting regulation. For example, we documented that the polyamine spermidine globally influences the generation of truncated mRNAs. In one case, we showed that high spermidine concentrations increased levels of RNA fragments derived from an mRNA encoding a spermidine import system, with a concomitant decrease in levels of the full-length mRNA. Collectively, our findings revealed new insight into transcription termination and uncovered an abundance of potential RNA regulators. Overall design: Identification of 3′ ends by 3′RNA-seq (Term-seq) experiment

转录终止（Transcription termination）是一类不可或缺且高度动态的生物学过程，可响应多样分子信号以调控基因表达。然而，此前仅在模式细菌中对转录终止的基因组定位、分子机制与调控效应开展了系统性深入研究。
本研究采用互补式RNA测序（RNA-seq）策略，对莱姆病致病病原体疏螺旋体属伯氏疏螺旋体（Borrelia burgdorferi）的转录组进行RNA末端定位。通过以单核苷酸分辨率系统性定位伯氏疏螺旋体的RNA末端，我们解析了其复杂的基因排布与操纵子结构，并定位了非翻译区（untranslated regions, UTRs）与小RNA（small RNAs, sRNAs）。
我们通过实验验证了伯氏疏螺旋体转录终止的多种模式，并将研究结果与大肠杆菌（E. coli）、铜绿假单胞菌（P. aeruginosa）及枯草芽孢杆菌（B. subtilis）中的已有观测结果进行了对比。我们发现，伯氏疏螺旋体63%的RNA 3′末端定位于开放阅读框（open reading frames, ORFs）的上游或内部，这提示存在全新的调控机制。
Northern印迹（Northern analysis）实验证实，编码伯氏疏螺旋体独特感染周期相关基因产物的mRNA可产生稳定的5′端衍生RNA片段。我们推测这类RNA源于提前终止事件与调控过程，包括顺式作用调控形式。例如，我们记录到多胺亚精胺（spermidine）会全局性影响截短型mRNA的产生。在一例具体案例中，我们证实高浓度亚精胺会提升编码亚精胺转运系统的mRNA所产生的RNA片段水平，同时伴随全长mRNA水平的显著下降。
综上，本研究为转录终止机制提供了全新认知，并揭示了大量潜在的RNA调控因子。
整体实验设计：通过3′RNA测序（3′RNA-seq，Term-seq）实验鉴定RNA 3′末端。