Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RIP-Seq]. Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RIP-Seq]

Bacterial small non-coding RNAs (sRNAs) play post-transcriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV crosslinking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the P. aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5’ UTRs and sRNAs, and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that sRNAs association with Hfq is primarily a function of their expression levels, strongly supporting that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of post-transcriptional regulation in P. aeruginosa. Overall design: To detect the binding sites for P. aeruginosa Hfq between different conditions we collected three biological replicates of P. aeruginosa PAO1 hfq::3xFLAG strain from planktonic (OD600 = 2.0) and biofilm (48 hour-old on LB agar). Half of each replicate culture was irradiated with UV light (254 nm, 800 mJ/cm2) (+XL), while the other half was left untreated (-XL). Bacteria were lysed and the FLAG-tagged protein was immunoprecipitated using a monoclonal anti-FLAG antibody. The samples were treated with benzonase nuclease, calf intestine phosphorylase, and polynucleotide kinase in the presence of radioactive gamma-ATP. Samples were separated with SDS-PAGE followed by transfer to nitrocellulose membranes. Radioactively labelled RNA-protein complexes were eluted from membranes and treated with Proteinase K. Purified RNA was used as input for library preparation using the NEB Next Small RNA Library kit. Besides, total RNA was purified from both planktonic and biofilm conditions and the high-throughput sequencing was performed at Core Unit Systems Medicine, University Hospital of Würzburg, Germany.

细菌小型非编码RNA（small non-coding RNAs, sRNAs）在细胞响应环境信号变化以及适应恶劣环境的过程中发挥转录后调控功能。通常情况下，RNA结合蛋白Hfq可辅助sRNAs结合靶标mRNA，以调控其翻译过程，并依据生理状态重塑全局RNA库。本研究通过联合应用体内紫外交联免疫沉淀结合高通量测序（CLIP-seq）与总RNA-seq，发现铜绿假单胞菌（P. aeruginosa）的Hfq在浮游态与生物膜态条件下，可结合其转录组的不同区域。本研究采用的分析方法显示，铜绿假单胞菌Hfq优先结合mRNA 5'非翻译区（5' untranslated region, 5' UTR）及sRNAs上的AAN三联基序重复序列，以及ρ非依赖型终止子处的富U序列。进一步的转录组分析表明，sRNAs与Hfq的结合主要由其自身表达水平决定，这有力支持了"Hfq结合RNA池是通过RNA浓度驱动的在Hfq上结合与解离的循环实现动态平衡"这一结论。综上，我们的联合CLIP-seq与总RNA-seq研究策略，揭示了铜绿假单胞菌中sRNAs与Hfq的条件性结合，这是该菌转录后调控的全新维度。整体实验设计：为检测不同条件下铜绿假单胞菌Hfq的结合位点，我们从浮游态（OD600=2.0）与LB琼脂上培养48小时的生物膜态样本中，收集了铜绿假单胞菌PAO1 hfq::3xFLAG标记菌株的三个生物学重复。每个重复培养物的一半经紫外照射（254nm，800mJ/cm²，记为+XL组），另一半不做处理（记为-XL组）。裂解细菌后，使用单克隆抗FLAG抗体免疫沉淀FLAG标记的Hfq蛋白。随后将样品用苯酶核酸酶、小牛肠磷酸酶及多核苷酸激酶处理，并添加放射性γ-ATP。样品经SDS-PAGE分离后转印至硝酸纤维素膜，从膜上洗脱放射性标记的RNA-蛋白复合物，再用蛋白酶K处理。纯化得到的RNA作为起始材料，使用NEB Next小RNA文库制备试剂盒构建测序文库。此外，我们从浮游态和生物膜态样本中纯化总RNA，高通量测序工作在德国维尔茨堡大学医院系统医学核心实验室完成。