CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

Abstract

Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5= untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3= to 5= exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNAseq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5= fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. IMPORTANCECsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity.

Airpnp: Auto- and Integrated Regulation of Polynucleotide Phosphorylase

“…While the benefits of a complex associating an endoribonuclease, a 3= exonuclease, and a helicase are evident and have been shown experimentally in E. coli, the role of enolase in the degradosome has never been very clear. The hypothesis that it may serve to link RNA degradation to carbon metabolism is reinforced by the observation by Park et al in this issue (14) that CsrA, which is intrinsically linked to carbon metabolism, is a repressor of pnp translation. In Deinococcus radiodurans, a different RNA-degrading complex exists in which a noncoding RNA called Y-RNA tethers PNPase to an ortholog of the Ro autoantigen called Rsr.…”

“…Translation inhibition by CsrA is totally dependent on prior cleavage of the stem-loop by RNase III. Although it is not directly addressed in the paper by Park et al (14), one can imagine that the regulation of pnp gene expression in the cell is influenced by factors that affect CsrA levels, such as those that impact the expression of the csrA gene itself (16) or that of CsrB and CsrC, two small (sponge) RNAs that contain multiple CsrA binding sites and titrate the protein (17).…”

“…If this were not complicated enough, in this issue of the Journal of Bacteriology, Babitzke and colleagues have provided evidence for an additional layer of translational control through CsrA, integrated into the PNPase autoregulatory pathway (14). CsrA is a global regulator of quorum sensing, virulence, motility, biofilm formation, and carbon metabolism in E. coli (15).…”

The small RNA SraG participates in PNPase homeostasis

“…Cleavage by RNase III is also required for CsrA to repress pnp translation (Park et al 2015). On the other hand, when RNase III is absent, PNPase can inhibit its own translation by competing with the binding of S1 on its own mRNA (Carzaniga et al 2015).…”

“…The destabilizing effect of PNPase on its own mRNA is also less efficient at low temperatures, leading to a decrease in repression and an increase in the expression level (Beran and Simons 2001;Mathy et al 2001). In addition, CsrA binds two sites in the pnp 5 ′ UTR, after its processing by RNase III and PNPase, where one CsrA site overlaps the pnp ShineDalgarno sequence (SD) such that CsrA binding inhibits pnp translation (Park et al 2015).…”