Discovery of a global translation activator in Pseudomonas aeruginosa [CLIP-Seq]
We identified PhaF as an RNA binding protein in Pseudomonas aeruginosa. In order to identify the different target transcripts of PhaF, we carried out the CLIP (Crosslinking and immunoprecipitation) and CLAP-Seq (Covalent linkage affinity purification) approaches, which utilizes UV irradiation to crosslink PhaF with the transcripts that are in close vicinity to it. For CLIP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal VSVG tag on PhaF. In order to determine the importance of the C-terminal domain (CTD) of PhaF on RNA binding, we used PAO1?phaF strains carrying plasmids that express a C-terminal VSVG tagged version of PhaF-CTD (pPhaF-CTD-V), along with a control strain that carries the same plasmid (pPhaF-CTD) but without the VSVG tag. For CLAP-Seq experiments, we used the Pseudomonas aeruginosa PAO1 strain along with a derivative of PAO1 that harbors a C-terminal Halo tag on PhaF. All the strains were subjected to UV irradiation, lysed, immunoprecipitated using either anti-VSVG-antibody coated beads or Magne Halo tagged beads. Following immunoprecipitation, the RNAs were purified. We also purified Total (Tot) RNA (samples collected before immunoprecipitation) from the PAO1 strains harboring the C-terminal VSVG tag on PhaF. The purified RNA samples were converted to cDNA libraries, which were subsequently sequenced using the Illumina NextSeq. The sequences were mapped to the genome to identify the target transcripts. In a separate series of experiments we also carried out RNA-Seq to identify the genes that are differentially regulated using Pseudomonas aeruginosa PAO1 and PAO1?phaF strains. The isolated RNA was sent to SeqCenter (Pittsburgh, PA) and the sequences obtained were mapped to the genome and DESeq analysis was performed. Overall design: Strains used for CLIP-Seq were PAO1 and a derivative of PAO1 that harbors a C-terminal VSVG tag on PhaF along with PAO1 ?phaF strains carrying plasmids that express a C-terminal VSVG tagged version of PhaF-CTD (pPhaF-CTD-V), and a control strain that carries the same plasmid (pPhaF-CTD) but without the VSVG tag. For CLAP-Seq we used PAO1 strain along with a derivative of PAO1 that harbors a C-terminal Halo tag on PhaF. In both CLIP-Seq and CLAP-Seq experiments, LB grown overnight cultures were back-diluted to an OD600 of 0.01. The strains were grown with aeration at 37°C to an OD600 of 2.0. For expressing the CTD of PhaF (either pPhaF-CTD or pPhaF-CTD-V, cloned in the pPSV38 plasmid background), 2mM IPTG was added to the culture to induce expression along with 30 µg/ml of Gentamicin.