Targeted degradation of host messenger RNA by a bacterial pathogen [CLIP-seq]

Post-transcriptional processing and modification of messenger RNA (mRNA) regulates gene expression in eukaryotes by dictating the stability, localisation and translation of newly synthesised transcripts 1. These epitranscriptomic events are coordinated by an extensive network of RNA-binding proteins 2,3. Here we observed that the bacterial pathogen, Legionella pneumophila, caused the selective degradation of host cellular mRNAs encoding factors involved in glycolysis and related metabolic pathways. Screening of a library of L. pneumophila deletion mutant strains revealed a single effector protein, LegC4, that mediated the post-transcriptional degradation of host mRNAs encoding key glycolytic enzymes, thereby suppressing host glycolysis during infection. Using CLIP-seq and complementary methods, we observed that LegC4 bound to mature processed host mRNA recognising a guanine (G)-rich motif that was overrepresented within mRNAs targeted for degradation. In vitro activity assays showed that LegC4 harboured intrinsic RNase activity and structural determination of a catalytically inactive mutant of LegC4 in complex with single-stranded RNA revealed a unique RNA-binding domain 4. The selective binding and degradation of host mRNA by LegC4 reveals a previously undescribed mechanism of bacterial effector protein activity targeting the host epitranscriptome. Overall design: To isolate the host RNA-binding targets of Legionella effector protein LegC4, THP-1 macrophages were infected with a Legionella pneumophila 130b strain (deltalegC4) carrying a p4HALegC4-protein expression plasmid or p4HA control plasmid. We then performed cross-linking immunoprecipitation (CLIP) using an anti-HA antibody and RNA-seq of samples isolated by LegC4 (LegC4 IP) versus the control (Control IP) across four independent experiments.