Bacterial/archaeal/organellar polyadenylation

Abstract

Although the first poly(A) polymerase (PAP) was discovered in Escherichia coli in 1962, the study of polyadenylation in bacteria was largely ignored for the next 30 years. However, with the identification of the structural gene for E. coli PAP I in 1992, it became possible to analyze polyadenylation using both biochemical and genetic approaches. Subsequently, it has been shown that polyadenylation plays a multifunctional role in prokaryotic RNA metabolism. Although the bulk of our current understanding of prokaryotic polyadenylation comes from studies on E. coli, recent limited experiments with Cyanobacteria, organelles, and Archaea have widened our view on the diversity, complexity, and universality of the polyadenylation process. For example, the identification of polynucleotide phosphorylase (PNPase), a reversible phosphorolytic enzyme that is highly conserved in bacteria, as an additional PAP in E. coli caught everyone by surprise. In fact, PNPase has now been shown to be the source of post-transcriptional RNA modifications in a wide range of cells of prokaryotic origin including those that lack a eubacterial PAP homolog. Accordingly, the past few years have witnessed increased interest in the mechanism and role of post-transcriptional modifications in all species of prokaryotic origin. However, the fact that many of the poly(A) tails are very short and unstable as well as the presence of polynucleotide tails has posed significant technical challenges to the scientific community trying to unravel the mystery of polyadenylation in prokaryotes. This review discusses the current state of knowledge regarding polyadenylation and its functions in bacteria, organelles, and Archaea.

Keywords: Hfq; RNA degradation; poly(A) polymerase; polynucleotide phosphorylase.

Fig. 1

Addition of (A) poly(A) tails by PAP I and (B) polynucleotide tails by PNPase in E. coli. Since the equilibrium constant of the PNPase catalyzed reaction is close to one, the enzyme can work either degradatively or biosynthetically depending upon the availability of inorganic phosphate (Pi). High NDP and low Pi concentrations favor the biosynthetic reaction which generates untemplated polynucleotide tails. Low NDP and high Pi concentrations favor the exoribonucleolytic degradation of transcripts. N: any nucleotide.

Fig. 2

Poly(A) tail profiles in wild type and pcnB deletion strains of E. coli. Total RNA was processed for the poly(A) sizing assay as described by Mohanty et al. [55].

Fig. 3

Predicted secondary structures of various Rho-independent transcription terminators in E. coli. Sequencing of cDNAs copies of these four transcripts has confirmed the nature of each single-stranded 3’ extension [12,20,47].

Fig. 4

Hfq mediated polyadenylation by PAP I in E. coli. Recent studies suggest that Rho-independent transcription terminators in E. coli transcripts may serve as polyadenylation signals [12,20]. Hfq has been shown to preferentially bind to the base of A/U rich region of the terminator [12]. It has been hypothesized that Hfq in its hexameric form interacts with PAP I and PNPase to form a polyadenylation complex [12], which binds to the base of the stem-loop associated with the Rho-independent transcription terminator. Consequently, some or all of the A/U base pairs of the stem loop melt permitting PAP I to bind to the resulting single-stranded 3’ end and add poly(A) tails processively. The interaction is believed to help PAP I to compete the vast excess of 3’ → 5’ exoribonucleases in order to find its substrate and to also suppress PNPase’s biosynthetic activity [12]. Terminal 5’ triphosphates can be converted to 5’ phosphomonoesters by RppH [161], a requirement for polyadenylation in vitro [22]. However, this requirement has not been demonstrated in vivo. An endoribonuclease such as RNase E may access the transcript from the 5’ end at the same time. The 5’ phosphorylation status, which can affect RNase E activity, probably varies for individual transcripts [–164]. In addition, it is also possible that RNase E can access the substrate as part of the degradosome by binding to the poly(A) tail (Fig. 5B). Once PAP I dissociates, along with Hfq, PNPase can degrade the poly(A) tail in the 3’→5’ direction. Endonucleolytically derived decay intermediates are mostly degraded by 3’→5’ exoribonucleases such as PNPase, RNase II, and RNase R. However, some of the decay intermediates contain strong G/C rich secondary structures forcing PNPase to stall and possibly switch to a biosynthetic mode, thereby generating unstructured polynucleotide tails (Fig. 6). These tails either change the conformation of the substrate or provide necessary single-stranded region for either PNPase, RNase II, or RNase R to bind and complete the degradation process.

Fig. 5

Polyadenylation assisted RNA decay in E. coli. (A) Addition of poly(A) tails by PAP I to the 3’ ends of an RNA substrate provides the single stranded binding site for both PNPase and RNase II that initiate the degradation. While PNPase catalyzes both 3’→ 5’ phosphorolytic degradation in presence of in organic phosphate and 5’→ 3’ polymerization in presence of NDPs, RNase II can only degrade RNA hydrolytically in the 3’→ 5’ direction. Both the ribonucleases pause upon encountering a G/C rich secondary structure. PNPase either dissociates relatively quickly or reverses its activity to polymerize polynucleotide tails. Dissociation of PNPase may initiate multiple rounds of polymerization by PAP I. In contrast, RNase II remains bound to the base of the secondary structure thereby effectively blocking the binding of either PAP I or PNPase. (B) RNase E alone or as part of the multiprotein complex called the degradosome can bind A/U rich poly(A) and polynucleotide tails to initiate degradation of a potential substrate through endonucleolytic cleavage. A full-length polyadenylated RNA substrate may be degraded very fast [114] by direct or internal entry [164] resulting in very few steady-state polyadenylated RNA species. This type of RNase E entry to an RNA substrate has yet to be experimentally demonstrated. (C) Potential poly(A) binding proteins can block RNA decay in E. coli. Proteins such as CspE, Hfq and ribosomal protein S1 could bind to poly(A) or polynucleotide tails blocking endonucleolytic access by the RNase E-based degradosome through its PNPase moiety or direct exonucleolytic degradation by exoribonucleases such as RNase II, RNase R, and PNPase.

Fig. 6

Secondary structure of a polynucleotide tail that was cloned and sequenced from a pcnB transcript of E. coli, (257 nt, −3.0 KCal) [20]. Similar analysis of polynucleotide tails derived from a pnp transcript of S. antibioticus, (116 nt, −0.6 KCal) [165], an rpsD transcript of B. subtilis (56 nt, −4.2 KCal) [29], a psbA transcript from spinach chloroplast (177 nt, −2.4 KCal) [30], an rbcL transcript from Synechocystis (172 nt, −1.9 KCal), [31], and an exosome complex exonuclease 2 transcript from M. kandleri (124 nt, −2.5 KCal), [15] yielded identical results (data not shown). The secondary structures and energy level (total energy for all the stems in a structure) were obtained by using RNA STAR program [120].