The minimal ProQ protein of Neisseria meningitidis is a global RNA-binding protein that operates in parallel with Hfq [CLIP-Seq]

In this study binding sites for the RNA-binding protein ProQ was determined in Neisseria meninigitidis serogroup C strain 8013 To detect the binding sites for Neisseria meningitidis (N.meninigitidis) ProQ we collected two biological replicates of N.meninigitidis serogroup C strain 8013 where the C-terminus of the proQ gene was fused to a 3xFLAG tag. Half of both replicate cultures was irradiated with UV light (254 nm, 800 mJ/cm2) (+CL), while the other half was kept untreated (-CL). After lysis of the bacteria, the FLAG-tagged protein was immunoprecipitated using a commercial monoclonal anti-FLAG antibody. The samples were treated with benzonase nuclease, calf intestine phophorylase (CIP), and polynucleotide kinase (PNK) and labeled with radioactive gamma-ATP. After separating the samples by SDS-PAGE, the ProQ-RNA complexes were transfer to nitrocellulose membranes. Radioactively labelled complexes were cut out and 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.