Identification and functional characterization of pseudouridine in mRNAs and non-coding RNAs of the bacterial human pathogen Campylobacter jejuni

在所有的生命王国中，已经描述了100多种不同的RNA修饰。虽然大多数修饰存在于丰富的家务RNA中，如rRNA、tRNA和SnRNAs，但最近的全基因组方法也揭示了真核和古生物mRNAs的修饰。细菌mRNAs的修饰还没有报道。在这里，我们的目标是研究空肠弯曲杆菌的RNA修饰，这是目前人类细菌性胃肠炎最常见的原因，重点是伪尿苷(PU)。这种普遍保守的修饰RNA核苷是伪尿苷合成酶(PU)转录后产生的尿苷的异构体，是tRNA和rRNA中含量最丰富的修饰。利用伪序列，一种新的基于化学修饰的PU位点的反转录末端的深度测序的全球PU图谱方法，PU最近在人和酵母的mRNAs中被报道，但它的功能仍然是谜。胁迫条件下PU的增加表明它可能调节RNA的稳定性、结构，甚至编码潜力，因为人工引入的PU残基可以介导酵母中的无义抑制。利用无偏见的基因组学方法，我们的目标是在全球范围内对新出现的食源性致病菌空肠弯曲菌的转录本中的PU进行分析，并研究这种修饰在细菌mRNAs中的存在和功能。我们之前的基于RNA-seq的转录组分析显示，空肠弯曲菌有一个紧凑的转录输出和许多调控RNA，这表明转录后调控是基因表达调控的重要层面。利用免疫共沉淀结合RNA-seq(RIP-seq)对tRNA修饰的PUS TRUB的RNA底物进行全局研究，我们既可以丰富tRNAs，又可以确定几个mRNAs作为潜在的TRUB底物。我们的第一个空肠弯曲菌野生型(WT)RNA的伪序列成功地在tRNA和rRNA中检测到PU，并发现了几个具有潜在PU位点的候选mRNAs。通过将PUS酶的RIP-SEQ与在常规和应激条件下生长的WT和PUS突变株的伪序列相结合，我们将提供空肠弯曲菌PU位点和PUS共识基序的全球图谱。PU位点将通过引物延伸、RNA靶标与PUS酶的体外结合研究以及用纯化的PUS酶处理后体外合成的RNA的PU用薄层层析和质谱仪检测来验证。无铜点击化学将被用来在体外修饰或总RNA中将生物素或荧光团连接到PU上，以允许标记和纯化修饰RNA。我们将利用生化、分子生物学和遗传学的方法，研究PU在细菌mRNAs中的功能。将评估PU对选定候选mRNAs的RNA稳定性、结构和/或编码潜力的潜在变化。对空肠弯曲菌中PU的研究将为深入了解RNA修饰及其在真核生物中的功能提供潜在的转录后调控。

More than 100 different RNA modifications have been described in all kingdoms of life. While most modifications are found in abundant housekeeping RNAs such as rRNA, tRNA, and snRNAs, recent genome-wide approaches have also revealed modifications in eukaryotic and archaeal mRNAs. Modifications in bacterial mRNAs have not yet been reported. Here, we aim to study RNA modifications in Campylobacter jejuni, currently the most common cause of bacterial gastroenteritis in humans, with a focus on pseudouridine (PU). This universally-conserved modified RNA nucleoside is an isomer of uridine posttranscriptionally generated by pseudouridine synthases (PUS), and is the most abundant modification in tRNA and rRNA. Using Pseudo-seq, a novel method of global PU profiling based on deep sequencing of reverse transcription stops at chemically-modified PU sites, PU was recently reported in human and yeast mRNAs, yet its functions are still enigmatic. An increase in PU under stress conditions indicated it might modulate RNA stability, structure, or even coding potential, since artificially-introduced PU residues can mediate nonsense suppression in yeast. Using unbiased genomics approaches, we aim to globally profile PU in transcripts of the emerging food-borne pathogen C. jejuni and investigate the presence and function of this modification in bacterial mRNAs. Our previous RNA-seq-based transcriptome analysis revealed a compact transcriptional output and many regulatory RNAs in C. jejuni, indicating posttranscriptional regulation is an important layer of gene expression control. Using co-immunoprecipitation combined with RNA-seq (RIP-seq) to globally study RNA substrates of the tRNA-modifying PUS TruB, we could both enrich for tRNAs and identify several mRNAs as potential TruB substrates. Our first Pseudo-seq of C. jejuni wildtype (WT) RNA successfully detected PU in tRNA and rRNA and revealed several candidate mRNAs with potential PU sites. By combining RIP-seq of PUS enzymes with Pseudo-seq of WT and PUS mutant strains grown under routine and stress conditions, we will provide a global map of PU sites and PUS consensus motifs for C. jejuni. The PU sites will be validated by primer extension, in vitro binding studies of RNA targets and PUS enzymes, and detection of PU of in vitro-synthesized RNAs following treatment with purified PUS enzymes by thin-layer chromatography and mass spectrometry. Copper free click-chemistry will be used to attach biotin or fluorophores to PU in either in vitro-modified or total RNA to allow for labeling and purification of modified RNA. Using biochemical, molecular biology, and genetics methods, we will investigate the functions of PU in bacterial mRNAs. Potential changes in RNA stability, structure, and/or coding potential by PU for selected candidate mRNAs will be assessed. The study of PU in C. jejuni will provide insight into potential posttranscriptional regulation by RNA modifications and their function, also in eukaryotes.