Transcriptome mapping and Hfq RIP-seq of Neisseria meningitidis

Neisseria meningitidis is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experimental evidence that gene regulation as well as the expression of small non-coding RNAs (sRNAs) affect meningococcal virulence, the organization of its transcriptome, including in particular the biogenesis of sRNAs and their mode of action, is only poorly understood. Here, we addressed these issues using a combination of high-throughput technologies. We applied differential RNA-seq (dRNA-seq) to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8103. Our dRNA-seq analysis predicted 1,625 transcriptional start sites (TSS) including 65 non-coding RNA transcripts, of which 20 were further validated by Northern analysis. This allowed for the discovery of a novel CRISPR-associated sRNA with a Cas9-independent biogenesis. Genome-wide mapping of s 70-dependent and independent promoters revealed that classical Escherichia coli-like s70 promoter are absent in most of the protein coding genes in meningococci. The majority of the 706 primary TSSs (pTSSs) were associated with coding sequences, including 382 pTSS obtained for single genes and 240 pTSSs obtained for genes located in operons. By Hfq RNA immunoprecipitation sequencing (RIP-seq) we identified a large Hfq-centered post-transcriptional regulatory network comprising 24 sRNAs and 407 potential mRNA targets, and rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on sRNAs. We finally confirmed the interactions of two sRNAs and their cognate target mRNA in vivo. Both directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.The combination of both high-throughput approaches thus creates a compendium that not only provides a valuable data resource, but also allows for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx. Overall design: differential RNA-seq at two different growth phases; Hfq RIP-seq

脑膜炎奈瑟菌（Neisseria meningitidis）是一种人类共生菌群，偶尔可引发细菌性脑膜炎、败血症等危及生命的感染。尽管已有实验证据表明，基因调控与小非编码RNA（small non-coding RNAs, sRNAs）的表达均会影响脑膜炎球菌的毒力，但目前对其转录组的组织架构，尤其是小非编码RNA的生物发生过程及其作用模式，仍缺乏深入认知。本研究结合多种高通量技术手段，针对上述科学问题开展探究。我们采用差异RNA测序（differential RNA-seq, dRNA-seq）技术，绘制了脑膜炎奈瑟菌菌株8103的单核苷酸分辨率级初级转录组图谱。通过dRNA-seq分析，共预测得到1625个转录起始位点（transcriptional start sites, TSS），其中包含65个非编码RNA转录本，其中20个经Northern印迹分析（Northern analysis）得到验证。该图谱助力我们发现了一种新型的、不依赖Cas9的CRISPR相关小RNA（CRISPR-associated sRNA）。全基因组范围内的σ70依赖型与非依赖型启动子定位分析显示，脑膜炎球菌的多数蛋白编码基因中均不存在类似大肠杆菌（Escherichia coli）的经典σ70启动子。在706个初级转录起始位点（primary TSSs, pTSSs）中，绝大多数与编码序列相关，其中382个pTSS对应单个独立基因，240个pTSS对应操纵子（operons）内的基因簇。通过Hfq RNA免疫沉淀测序（RNA immunoprecipitation sequencing, RIP-seq），我们鉴定得到一个以Hfq为核心的大型转录后调控网络，该网络涵盖24个小非编码RNA与407个潜在mRNA靶标；利福平（rifampicin）稳定性实验证实，Hfq结合可显著提升小非编码RNA的稳定性。我们最终在体内验证了两种小非编码RNA与其同源靶标mRNA的相互作用，二者均可直接抑制编码甲基异柠檬酸裂解酶（methylisocitrate lyase）的prpB基因表达——该基因此前已被证实参与脑膜炎球菌在人类鼻咽部的定植过程。综上，本研究通过两种高通量技术构建的转录组编目不仅提供了极具价值的数据集资源，还增进了我们对脑膜炎球菌转录组组织架构与核糖调控机制的理解，其研究结果对阐释人类鼻咽部定植过程具有参考意义。整体实验设计：两种不同生长阶段的差异RNA测序；Hfq RIP-seq