Victoria Portnoy

RNA polyadenylation in Archaea : not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus

The addition of poly(A) tails to RNA is a phenomenon common to all organisms examined so far. No homologues of the known polyadenylating enzymes are found in Archaea and little is known concerning the mechanisms of messenger RNA degradation in these organisms. Hyperthermophiles of the genus Sulfolobus contain a protein complex with high similarity to the exosome, which is known to degrade RNA in eukaryotes. Halophilic Archaea, however, do not encode homologues of these eukaryotic exosome components. In this work, we analysed RNA polyadenylation and degradation in the archaea Sulfolobus solfataricus and Haloferax volcanii. No RNA polyadenylation was detected in the halophilic archaeon H. volcanii. However, RNA polynucleotidylation occurred in hyperthermophiles of the genus Sulfolobus and was mediated by the archaea exosome complex. Together, our results identify the first organism without RNA polyadenylation and show a polyadenylation activity of the archaea exosome.

Domain Analysis of the Chloroplast Polynucleotide Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteins

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events, including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an α-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. Phylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.

RNA polyadenylation and degradation in different Archaea; roles of the exosome and RNase R

Polyadenylation is a process common to almost all organisms. In eukaryotes, stable poly(A)-tails, important for mRNA stability and translation initiation, are added to the 3′ ends of most mRNAs. Contrarily, polyadenylation can stimulate RNA degradation, a phenomenon witnessed in prokaryotes, organelles and recently, for nucleus-encoded RNA as well. Polyadenylation takes place in hyperthermophilic archaea and is mediated by the archaeal exosome, but no RNA polyadenylation was detected in halophiles. Here, we analyzed polyadenylation in the third archaea group, the methanogens, in which some members contain genes encoding the exosome but others lack these genes. Polyadenylation was found in the methanogen, Methanopyrus kandleri, containing the exosome genes, but not in members which lack these genes. To explore how RNA is degraded in the absence of the exosome and without polyadenylation, we searched for the exoribonuclease that is involved in this process. No homologous proteins for any other known exoribonuclease were detected in this group. However, the halophilic archaea contain a gene homologous to the exoribonuclease RNase R. This ribonuclease is not able to degrade structured RNA better than PNPase. RNase R, which appears to be the only exoribonucleases in Haloferax volcanii, was found to be essential for viability.

RNA Polyadenylation in Prokaryotes and Organelles; Different Tails Tell Different Tales

The addition of poly(A) tails to RNA is a phenomenon common to almost all organisms examined as of today. In eukaryotes, a stable poly(A) tail is added to the 3′ end of most nuclear-encoded mRNAs. This process is important for mRNA stability and translation initiation. In addition, polyadenylation of nuclear-encoded transcripts in yeast was recently reported to promote RNA degradation. In prokaryotes and organelles, RNA molecules are polyadenylated as part of a polyadenylation-dependent RNA degradation mechanism. This process consists sequentially of endonucleolytic cleavage, addition of degradation-inducing poly(A)-rich sequences to these cleavage products, and exonucleolytic degradation. In spinach chloroplasts the latter two steps, polyadenylation and exonucleolytic degradation, are performed by a single phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase), while there is no equivalent to the E. coli poly(A)-polymerase enzyme. This was also found to be the case in cyanobacteria, a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, and also in several other bacteria. No RNA polyadenylation was detected in the halophilic archaea Haloferax volcanii, which lacks the exosome complex, or in yeast mitochondria, which lack PNPase. Unlike other organelles, mammalian mitochondrial transcripts are known to include stable poly(A) tails at their 3′ ends, much like the case of nuclear-encoded mRNA. However, recent data have revealed that in addition to full-length, stably polyadenylated transcripts, nonabundant, truncated, polyadenylated RNA fragments are present in human mitochondria. These results suggest that the polyadenylation-dependent RNA degradation pathway is present in human mitochondria together with the addition of stable poly(A) tails at the mature 3′ end. We describe a possible scenario illustrating the evolution of RNA polyadenylation and its related functions found in bacteria, archaea, organelles, and eukaryotes. Referee: Dr. David Stern, Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853.

Processing, degradation, and polyadenylation of chloroplast transcripts

In this chapter, we describe the major enzymes and characteristics of transcript 5′ and3′ end maturation, and polyadenylation-stimulated degradation. The picture which emerges isthat maturation and degradation share many prokaryotic features, vestiges of the chloroplast endosymbiontancestor. The major exoribonucleases are well-defined, being polynucleotide phosphorylase and RNaseII/R. The endonucleases include CSP41, with largely informatic evidence for homologs of prokaryoticRNases E, J, and III. The polyadenylation-stimulated degradation pathway, which occurs in most livingsystems, is a major player in chloroplast RNA degradation. We discuss known or potential rolesfor polynucleotide phosphorylase and a prokaryotic-type poly(A) polymerase. Finally, we discussnuclear mutations that affect RNA maturation and degradation, defining genes that are likely or knownto encode regulatory factors. Major questions for future research include how the ribonucleases,which are inherently nonspecific, interact with these specificity factors, and whether newly-discoverednoncoding RNAs in the chloroplast play any role in RNA metabolism.

Distinct activities of several RNase J proteins in methanogenic archaea

RNA degradation plays an important role in the control of gene expression in all domains of life, including Archaea. While analyzing RNA degradation in different archaea, we faced an interesting situation. The members of a group of methanogenic archaea, including Methanocaldococcus jannaschii, contain neither the archaeal exosome nor RNase II/R homologs. However, looking for potential ribonucleases revealed proteins related to the recently discovered ribonuclease RNase J. RNase J is unique among known ribonucleases because it may combine endo- and 5’→3’ exo-ribonucleolytic activities in a single polypeptide. Here, we report the characterization of the ribonuclease activities of three RNase J homologs encoded in the genome of the methanogenic archaeon Methanocaldococcus jannaschii. The analysis of the recombinant archaeal proteins purified from E. coli revealed an optimal activity at 60°C. Whereas mjRNase J1 and -J3 displayed exclusively 5’→3’ exonucleolytic activity, mjRNase J2 is an endonuclease with no apparent exonuclease activity. The exonucleolytic activity of both mjRNase J1 and -J3 is enhanced in molecules harboring monophosphate at the 5’ end. mjRNase J3, and to some extent mjRNase J2, degrade ssDNA. Together, these results reveal that in archaea lacking the exosome and RNase II/R, RNA and perhaps also DNA are possibly degraded by the coordinated activities of several RNase J proteins. Unlike bacteria, in archaea RNase J proteins provide separately the exo- and endonucleolytic activities that are probably essential for RNA degradation.

Mycoplasma gallisepticum as the first analyzed bacterium in which RNA is not polyadenylated

The addition of poly(A)-tails to RNA is a phenomenon common to almost all organisms. In addition to most eukaryotic mRNAs possessing a stable poly(A)-tail, RNA is polyadenylated as part of a degradation mechanism in prokaryotes, organelles, and the eukaryotic nucleus. To date, only very few systems have been described wherein RNA is metabolized without polyadenylation, including several archaea and yeast mitochondria. The minimal genome of the parasitic bacteria, Mycoplasma, does not encode homologs of any known polyadenylating enzyme. Here, we analyze polyadenylation in Mycoplasma gallisepticum. Our results suggest this organism as being the first described bacterium in which RNA is not polyadenylated.