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

超过100种不同的RNA修饰已经被描述在所有的生命王国中。虽然大多数修饰是在丰富的管家rna中发现的，如rRNA、tRNA和snrna，但最近的全基因组方法也揭示了真核生物和古细菌mrna的修饰。细菌mrna的修饰尚未被报道。在这里，我们的目标是研究空肠弯曲杆菌（Campylobacter jejuni）的RNA修饰，这是目前人类细菌性胃肠炎最常见的原因，重点是假尿嘧啶（PU）。这种普遍保守的修饰RNA核苷是由伪尿苷合成酶（pseudo - urridine synthases, pu）转录后产生的尿苷同分异构体，是tRNA和rRNA中最丰富的修饰。利用Pseudo-seq，一种基于化学修饰的PU位点逆转录停止深度测序的全球PU分析新方法，最近在人类和酵母mrna中报道了PU，但其功能仍然是谜。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。利用无偏倚的基因组学方法，我们的目标是在全球范围内分析新出现的食源性病原体空肠梭菌的转录本中的PU，并研究这种修饰在细菌mrna中的存在和功能。我们之前基于rna -seq的转录组分析显示，空肠c具有紧凑的转录输出和许多调控rna，表明转录后调控是基因表达控制的重要层。利用共免疫沉淀结合RNA-seq （RIP-seq）技术对trna修饰的PUS TruB的RNA底物进行全局研究，我们既可以富集trna，又可以鉴定出几种可能作为TruB底物的mrna。我们的第一个空肠C.野生型（WT） RNA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mrna。通过将PU酶的RIP-seq与常规和逆境条件下生长的WT和PU突变菌株的Pseudo-seq相结合，我们将提供一个全球的PU位点和PU共识基序图。这些PU位点将通过引物延伸、RNA靶点与PUS酶的体外结合研究、纯化的PUS酶处理后体外合成的RNA的PU检测（薄层色谱和质谱）进行验证。无铜点击化学将用于将生物素或荧光团附着在体外修饰或总RNA中的PU上，以便对修饰RNA进行标记和纯化。利用生物化学、分子生物学和遗传学的方法，我们将研究PU在细菌mrna中的功能。将评估PU对选定候选mrna的RNA稳定性、结构和/或编码潜力的潜在变化。空肠梭菌中PU的研究将为真核生物中潜在的RNA修饰及其功能的转录后调控提供新的思路。

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.