Shimyn Slomovic

Polyadenylation and Degradation of Human Mitochondrial RNA: the Prokaryotic Past Leaves Its Mark

ABSTRACT RNA polyadenylation serves a purpose in bacteria and organelles opposite from the role it plays in nuclear systems. The majority of nucleus-encoded transcripts are characterized by stable poly(A) tails at their mature 3′ ends, which are essential for stabilization and translation initiation. In contrast, in bacteria, chloroplasts, and plant mitochondria, polyadenylation is a transient feature which promotes RNA degradation. Surprisingly, in spite of their prokaryotic origin, human mitochondrial transcripts possess stable 3′-end poly(A) tails, akin to nucleus-encoded mRNAs. Here we asked whether human mitochondria retain truncated and transiently polyadenylated transcripts in addition to stable 3′-end poly(A) tails, which would be consistent with the preservation of the largely ubiquitous polyadenylation-dependent RNA degradation mechanisms of bacteria and organelles. To this end, using both molecular and bioinformatic methods, we sought and revealed numerous examples of such molecules, dispersed throughout the mitochondrial genome. The broad distribution but low abundance of these polyadenylated truncated transcripts strongly suggests that polyadenylation-dependent RNA degradation occurs in human mitochondria. The coexistence of this system with stable 3′-end polyadenylation, despite their seemingly opposite effects, is so far unprecedented in bacteria and other organelles.

Polyadenylation of ribosomal RNA in human cells

The addition of poly(A)-tails to RNA 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 nuclear-encoded mRNAs, but not to rRNAs. Contrarily, in prokaryotes and organelles, polyadenylation stimulates RNA degradation. Recently, polyadenylation of nuclear-encoded transcripts in yeast was reported to promote RNA degradation, demonstrating that polyadenylation can play a double-edged role for RNA of nuclear origin. Here we asked whether in human cells ribosomal RNA can undergo polyadenylation. Using both molecular and bioinformatic approaches, we detected non-abundant polyadenylated transcripts of the 18S and 28S rRNAs. Interestingly, many of the post-transcriptionally added tails were composed of heteropolymeric poly(A)-rich sequences containing the other nucleotides in addition to adenosine. These polyadenylated RNA fragments are most likely degradation intermediates, as primer extension (PE) analysis revealed the presence of distal fragmented molecules, some of which matched the polyadenylation sites of the proximal cleavage products revealed by oligo(dT) and circled RT–PCR. These results suggest the presence of a mechanism to degrade ribosomal RNAs in human cells, that possibly initiates with endonucleolytic cleavages and involves the addition of poly(A) or poly(A)-rich tails to truncated transcripts, similar to that which operates in prokaryotes and organelles.

Dis3-like 1: a novel exoribonuclease associated with the human exosome.

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine‐subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome‐associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity‐purified human exosome complexes identified a novel exosome‐associated exoribonuclease, human Dis3‐like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co‐immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA‐mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)‐tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome‐associated exoribonuclease in the cytoplasm of human cells.

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.

Addition of poly(A) and poly(A)-rich tails during RNA degradation in the cytoplasm of human cells

Polyadenylation of RNA is a posttranscriptional modification that can play two somewhat opposite roles: stable polyadenylation of RNA encoded in the nuclear genomes of eukaryote cells contributes to nuclear export, translation initiation, and possibly transcript longevity as well. Conversely, transient polyadenylation targets RNA molecules to rapid exonucleolytic degradation. The latter role has been shown to take place in prokaryotes and organelles, as well as the nucleus of eukaryotic cells. Here we present evidence of hetero- and homopolymeric adenylation of truncated RNA molecules within the cytoplasm of human cells. RNAi-mediated silencing of the major RNA decay machinery of the cell resulted in the accumulation of these polyadenylated RNA fragments, indicating that they are degradation intermediates. Together, these results suggest that a mechanism of RNA decay, involving transient polyadenylation, is present in the cytoplasm of human cells.

Exonucleases and endonucleases involved in polyadenylation‐ assisted RNA decay

RNA polyadenylation occurs in most forms of life, excluding a small number of biological systems. This posttranscriptional modification undertakes two roles, both of which influence the stability of the polyadenylated transcript. One is associated with the mature 3′ ends of nucleus‐encoded mRNAs in eukaryotic cells and is important for nuclear exit, translatability, and longevity. The second form of RNA polyadenylation assumes an almost opposite role; it is termed ‘transient’ and serves to mediate the degradation of RNA. Poly(A)‐assisted RNA decay pathways were once thought to occur only in prokaryotes/organelles but are now known to be a common phenomenon, present in bacteria, organelles, archaea, and the nucleus and cytoplasm of eukaryotic cells, regardless of the fact that in some of these systems, stable polyadenylation exists as well. This article will summarize the current knowledge of polyadenylation and degradation factors involved in poly(A)‐assisted RNA decay in the domains of life, focusing mainly on that which occurs in prokaryotes and organelles. In addition, it will offer an evolutionary view of the development of RNA polyadenylation and degradation and the cellular machinery that is involved. WIREs RNA 2011 2 106–123 DOI: 10.1002/wrna.45