Post-transcriptional regulation of the MiaA prenyl transferase by CsrA and the small RNA CsrB inE. coli

Abstract

ABSTRACTTo determine the role of small RNAs (sRNAs) in the regulation ofmiaA, we constructed a chromosomalmiaA-lacZtranslational fusion driven by the arabinose responsive PBADpromoter and used it to screen against anEscherichia colismall RNA library (containing small RNAs driven by the IPTG inducible PLacpromoter). Our genetic screen and quantitative β-galactosidase assays identified CsrB and its cognate protein CsrA as potential regulators ofmiaAexpression inEscherichia coli. Consistent with our hypothesis that CsrA regulates MiaA post-transcriptional gene expression through binding to the MiaA mRNA 5’ UTR, and CsrB binds and regulates MiaA post-transcriptional gene expression through sequestration of CsrA levels, a deletion ofcsrAsignificantly reduced expression of the reporter fusion as well as reducingmiaAmRNA levels. These results suggest under conditions where CsrA is inhibited, MiaA translation and thus MiaA-dependent tRNA modification may be limiting.IMPORTANCEWe previously demonstrated a role for the i6A modification in the tuning of transcripts for several stress response genes inE. coli. The i6A tRNA modification is catalyzed by the tRNA prenyl transferase encoded by themiaAgene. We set out to identify posttranscriptional regulators of the enzyme necessary for the catalysis of i6A, MiaA, to further understand factors influencing i6A levels in the cell. We identified the CsrA RNA Binding Protein, the CsrB Small RNA, and RNA Degradosome enzymes: RNaseE and PNPase as regulators ofmiaAexpressioin at the post-transcriptional level. Identifying these post-transcripitonal regulators ofmiaAwill help us understand factors influencing i6A levels and may guide future investigations into RNA modifications with regulatory effects on the transcriptome.