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

Abstract

To determine the role of small RNAs (sRNAs) in the regulation of miaA, we constructed a chromosomal miaA-lacZ translational fusion driven by the arabinose responsive PBAD promoter and used it to screen against an Escherichia coli small RNA library (containing small RNAs driven by the IPTG inducible PLac promoter). Our genetic screen and quantitative {beta}-galactosidase assays identified CsrB and its cognate protein CsrA as potential regulators of miaA expression in Escherichia 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 of csrA significantly reduced expression of the reporter fusion as well as reducing miaA mRNA 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 in E. coli. The i6A tRNA modification is catalyzed by the tRNA prenyl transferase encoded by the miaA gene. 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 of miaA expressioin at the post-transcriptional level. Identifying these post-transcripitonal regulators of miaA will help us understand factors influencing i6A levels and may guide future investigations into RNA modifications with regulatory effects on the transcriptome.