Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RNA-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 disentangle differences in gene expression from those in protein binding, we performed RNA-seq for an ad hoc normalization of CLIP-seq

细菌小分子非编码RNA（small non-coding RNAs, sRNAs）在细胞响应环境变化及适应严苛环境的过程中，发挥转录后调控功能。通常而言，RNA结合蛋白Hfq可协助sRNAs与靶标mRNA结合，以调控其翻译过程，并依据细胞生理状态重塑全局RNA池。
本研究结合体内紫外交联免疫沉淀-高通量测序（CLIP-seq）与总RNA测序（total RNA-seq）技术，证实铜绿假单胞菌（P. aeruginosa）转录组在浮游态与生物膜态条件下，Hfq的结合区域存在显著差异。
实验结果显示，铜绿假单胞菌的Hfq优先结合mRNA 5'非翻译区（5' untranslated region, 5' UTR）与sRNAs中的AAN三联基序重复序列，以及ρ非依赖型转录终止子（rho-independent terminators）处的富U序列。
进一步的转录组分析表明，sRNAs与Hfq的结合主要取决于其自身的表达水平，这有力证实了Hfq结合RNA池的稳态由RNA浓度驱动的结合-解离循环所维持。
综上，本研究结合CLIP-seq与总RNA-seq的实验策略，揭示了铜绿假单胞菌中Hfq与sRNAs的条件性结合模式，这是该菌转录后调控的全新维度。
实验设计：为区分基因表达差异与蛋白结合差异，我们通过RNA-seq为CLIP-seq提供定制化的标准化参照。