Co-immunoprecipitation of the small DUF1127 protein RSP_6037xFLAG and pRK0557_3xFLAG for RNA interaction

Many protein domains are conserved among different classes and are found in numerous species, but their function remains obscure. Data bases list more than 17,000 DUF1127 proteins but to date no biological function could be assigned to any of these proteins. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in the maturation of the CcsR sRNAs that are processed from the 3´ UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 also affects the abundance of several other sRNAs but also of tRNAs, rRNAs and several mRNAs. Co-immunoprecipitation revealed direct binding of CcaF1 to many cellular RNAs of different type and an effect of CcaF1 on the stability of selected RNAs was demonstrated. Thus, the widely distributed DUF1127 domain can mediate RNA-binding and affect stability of its binding partners and consequently the bacterial transcriptome. Protein-RNA interaction was analyzed by co-immunoprecipitation. The strains Rhodobacter sphaeroides 2.4.1 pRK6037_3xFLAG (control pRK6037) and pRK0557_3xFLAG (control pRK0557) were used. RNA was isolated and sequenced.

诸多蛋白质结构域在不同类群间高度保守，且广泛存在于众多物种中，但其具体生物学功能仍未明确。目前数据库中已收录超过17000种DUF1127家族蛋白质，但截至当前，尚未有任何一种该类蛋白质被明确赋予生物学功能。本研究证实，来自α-变形菌纲（Alphaproteobacteria）球形红杆菌（Rhodobacter sphaeroides）的71个氨基酸长度的DUF1127家族蛋白质CcaF1，参与CcsR小RNA（small RNA, sRNA）的成熟过程——这些CcsR小RNA由ccaF信使RNA（messenger RNA, mRNA）的3'非翻译区（3' untranslated region, 3' UTR）加工而来，且在氧化应激防御中发挥作用。此外，CcaF1还可影响多种其他小RNA、转运RNA（transfer RNA, tRNA）、核糖体RNA（ribosomal RNA, rRNA）以及部分信使RNA的表达丰度。免疫共沉淀（Co-immunoprecipitation）实验结果显示，CcaF1可直接结合多种不同类型的细胞RNA，同时本研究也证实了CcaF1对特定RNA分子稳定性的调控作用。由此可见，分布广泛的DUF1127结构域可介导RNA结合，并通过调控其结合靶标的稳定性，进而影响细菌的转录组（transcriptome）。本研究通过免疫共沉淀技术分析了蛋白质-RNA相互作用。实验所用菌株为球形红杆菌（Rhodobacter sphaeroides）2.4.1 pRK6037_3xFLAG（对照为pRK6037）以及pRK0557_3xFLAG（对照为pRK0557）。实验中对RNA进行了提取与测序。