Polynucleotide phosphorylase: Not merely an RNase but a pivotal post-transcriptional regulator

Abstract

Almost 60 years ago, Severo Ochoa was awarded the Nobel Prize in Physiology or Medicine for his discovery of the enzymatic synthesis of RNA by polynucleotide phosphorylase (PNPase). Although this discovery provided an important tool for deciphering the genetic code, subsequent work revealed that the predominant function of PNPase in bacteria and eukaryotes is catalyzing the reverse reaction, i.e., the release of ribonucleotides from RNA. PNPase has a crucial role in RNA metabolism in bacteria and eukaryotes mainly through its roles in processing and degrading RNAs, but additional functions in RNA metabolism have recently been reported for this enzyme. Here, we discuss these established and noncanonical functions for PNPase and the possibility that the major impact of PNPase on cell physiology is through its unorthodox roles.

Fig 1. Domains and structure of PNPase.

(A) Domain organization of PNPase, using the C. crescentus PNPase crystal structure as a reference [44]. The complete PNPase trimer viewed from the (B) top and (C) side, with domains colored the same as in (A). (D) A cut-away view of the RNA-bound trimer interior of the PNPase trimer bound to an RNA substrate (orange). Ball and stick structures depict nitrogen atoms as blue and oxygen atoms as red. Inset panels illustrate (i) the interactions between the GSGG loop of each KH domain with the RNA backbone, (ii) base stacking interactions between phenylalanine residues of the FFRR loops and several RNA bases, and (iii) the active site residues that bind phosphate and the metal cofactor. PNPase, polynucleotide phosphorylase.

Fig 2. The numerous roles performed by PNPase.

Schematic showing the functions performed by PNPase in bacteria and mitochondria. (A) In E. coli and other gram-negative bacteria, PNPase functions as part of the RNA degradosome in bulk mRNA decay but also operates independently of this machine to process tRNAs and rRNAs, add polynucleotide tails to RNAs, and modulate sRNA stability. PNPase binds Hfq-bound sRNAs but only degrades unbound sRNAs, which impacts both positive and negative sRNA-mediated gene regulation in E. coli. In Deinococcus radiodurans, PNPase forms a complex with Rsr mediated via Y-RNAs that degrades misfolded rRNAs. (B) The locations and functions of hPNPase are controversial, but under normal cellular conditions, hPNPase is mainly localized to the IMS where it is able to facilitate translocation of 5S rRNA, RNase P RNA, and possibly RNase MRP RNA into the mitochondrial matrix. Within the mitochondrial matrix, hPNPase degrades mitochondrial RNA and plays a role in mitochondrial DNA maintenance. Release into the cytoplasm upon overexpression or permeabilization of the mitochondrial outer membrane during apoptosis leads to the decay of mRNAs and polyadenylated noncoding RNAs. hfq, host factor for phage Q_β; hPNPase, human PNPase; IMS, inner membrane space; MRP, mitochondrial RNA processing; mtRNA, mitochondrial RNA; PNPase, polynucleotide phosphorylase; Rsr, Ro sixty-related protein; sRNA, small regulatory RNA; SUV3, suppressor of Var 1, 3.