Cis and trans acting factors that regulate polynucleotide phosphorylase expression in Escherichia coli

The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) post-transcriptionally regulates its own expression at the level of mRNA stability and translatability. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double strand cleavage about in the middle of a long stem-loop in the 5'-UTR. This generates a double stranded stem composed of a small RNA with a 3'-OH overhang paired to the new 5'-end of the transcript. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The first one maintains that PNPase impedes translation of the RNase III processed pnp mRNA thus exposing the transcript to degradative pathways. According to the second model, PNPase would simply destroy a stabilizing double stranded structure at the 5'-end of pnp mRNA by degrading a small RNA generated by RNase III endonucleolytic cleavage. We created several mutations in the 5'-untranslated region in order to identify cis-acting determinants for PNPase autoregulation. In addition we analyzed the role of RNase E, the major endoribonuclease implicated in the degradation of mRNAs, in the destabilization of pnp mRNA. Overall, our results show that the removal of the double stranded stem at the 5'-end of pnp mRNA is necessary and sufficient to destabilize the transcript whereas the putative translational control by PNPase does not seem to play a role in autoregulation. Moreover, the native pnp mRNA with its long 5'-UTR appears to be stable and translatable whereas the processed transcript devoid of its 5'-double strand structure is poorly, if at all, translated and is rapidly degraded by RNase E. It thus appears that the role of PNPase in autoregulation is to simply remove the small RNA that both allows translation and prevents degradation of the RNase III-matured transcript. 1. Robert-Le Meur, M. and C. Portier. 1992. E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism. EMBO J. 11:2633-2641. 2. Robert-Le Meur, M. and C. Portier. 1994. Polynucleotide phosphorylase of Escherichia coli induces the degradation of its RNase III processed messenger by preventing its translation. Nucleic Acids. Res. 22:397-403. 3. Jarrige, A. C., Mathy, N., and Portier, C. (2001) PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader. EMBO J 20: 6845-6855.