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

人类病原体空肠弯曲菌 mRNA 和非编码 RNA 中假尿苷的鉴定和功能表征

• 批准号: 277446585
• 负责人: Professorin Dr. Cynthia Mira Sharma
• 金额: --
• 依托单位: Institut für Molekulare Infektionsbiologie (IMIB)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277446585?language=en
• 关键词: Identification; functional; characterization; pseudouridine; mRNAs

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.

在所有生命王国中，已经描述了100多种不同的RNA修饰。虽然大多数修饰在大量的管家RNA如rRNA、tRNA和snRNA中发现，但最近的全基因组方法也揭示了真核生物和古细菌mRNA中的修饰。细菌mRNA的修饰尚未报道。在这里，我们的目标是研究空肠弯曲菌的RNA修饰，目前是人类细菌性胃肠炎最常见的原因，重点是假尿苷（PU）。这种普遍保守的修饰的RNA核苷是由假尿苷酶（PUS）转录后产生的尿苷的异构体，并且是tRNA和rRNA中最丰富的修饰。使用Pseudo-seq，一种基于逆转录终止于化学修饰的PU位点的深度测序的全局PU分析的新方法，最近在人类和酵母mRNA中报道了PU，但其功能仍然是谜。压力条件下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。使用无偏基因组学方法，我们的目标是在全球范围内分析新兴食源性病原体C的转录本中的PU。jejuni，并研究这种修饰在细菌mRNA中的存在和功能。我们先前基于RNA-seq的转录组分析揭示了C.空肠，表明转录后调控是基因表达控制的重要层。使用免疫共沉淀结合RNA-seq（RIP-seq）来全面研究tRNA修饰的PUS TruB的RNA底物，我们既可以富集tRNA，又可以鉴定出几种mRNA作为潜在的TruB底物。我们的第一个C.空肠野生型（WT）RNA成功地检测到tRNA和rRNA中的PU，并揭示了几个具有潜在PU位点的候选mRNA。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Pseudo-seq相结合，我们将提供C.空肠。将通过引物延伸、RNA靶标和PUS酶的体外结合研究以及通过薄层色谱法和质谱法检测纯化PUS酶处理后体外合成RNA的PU来验证PU位点。无铜点击化学将用于将生物素或荧光团连接到体外修饰的或总RNA中的PU，以允许标记和纯化修饰的RNA。利用生物化学、分子生物学和遗传学方法，我们将研究PU在细菌mRNA中的功能。将通过PU评估选定候选mRNA的RNA稳定性、结构和/或编码潜力的潜在变化。对C. jejuni将提供深入了解潜在的转录后调控的RNA修饰及其功能，也在真核生物。