Transgenesis of mammalian PABP reveals mRNA polyadenylation as a general stress response mechanism in bacteria

Area of Science:

• Molecular Biology
• Bacterial Genetics
• RNA Metabolism

Background:

• In eukaryotes, mRNA 3'-polyadenylation recruits poly(A) binding protein (PABP) for stabilization.
• Conversely, in bacteria, polyadenylation typically targets mRNAs for degradation by 3'-exonucleases like PNPase.

Purpose of the Study:

• To investigate the role of mammalian nuclear poly(A) binding protein (PABPN1) in bacterial mRNA fate.
• To determine if PABPN1 can alter the function of mRNA polyadenylation in bacteria, contrasting with its known eukaryotic role.

Main Methods:

• Trans-expression of human PABPN1 in *Escherichia coli* (both chromosomally and extra-chromosomally).
• Assessment of polyadenylated mRNA stability and half-lives in the presence of PABPN1.
• Analysis of 3'-exonuclease PNPase association with polyadenylated mRNAs.
• Phenotypic analysis including plasmid copy number and cellular stress tolerance.
• Genome-wide RNA sequencing (RNA-seq) to identify affected transcripts.

Main Results:

• PABPN1 expression, but not a mutant form, stabilized polyadenylated mRNAs in *E. coli*, increasing their half-lives.
• PABPN1 prevented PNPase binding to poly(A)-tailed mRNAs, inhibiting degradation.
• PABPN1 trans-expression mimicked the effect of a *pcnB* mutation (encoding poly(A) polymerase I) and affected plasmid copy number.
• RNA-seq revealed a general upregulation of polyadenylated mRNAs upon PABPN1 expression, particularly those involved in stress response.
• PABPN1 expression or *pcnB* mutation conferred cellular tolerance to various stresses.

Conclusions:

• mRNA 3'-polyadenylation functions as a general stress response mechanism in *E. coli*.
• The bacterial mRNA fate can be modulated by expressing a heterologous poly(A) binding protein.
• PABPN1 acts as a stabilizer of polyadenylated mRNAs in bacteria, counteracting the typical degradation pathway.