The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements.

HeLa cytoplasmic extracts contain both 3'-5' and 5'-3' exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modified RNAs. These data indicate that 3'-5' exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for efficient 3'-5' exonucleolytic decay. Furthermore, 3'-5' exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE-containing mRNA turnover.     

Genetic and biochemical characterization of Drosophila Snipper: A promiscuous member of the metazoan 3'hExo/ERI-1 family of 3' to 5' exonucleases

The DnaQ-H family exonuclease Snipper (Snp) is a 33-kDa Drosophila melanogaster homolog of 3'hExo and ERI-1, exoribonucleases implicated in the degradation of histone mRNA in mammals and in the negative regulation of RNA interference (RNAi) in Caenorhabditis elegans, respectively. In metazoans, Snp, Exod1, 3'hExo, ERI-1, and the prpip nucleases define a new subclass of structure-specific 3'-5' exonucleases that bind and degrade double-stranded RNA and/or DNA substrates with 3' overhangs of 2-5 nucleotides (nt) in the presence of Mg2+ with no apparent sequence specificity. These nucleases are also capable of degrading linear substrates. Snp efficiently degrades structured RNA and DNA substrates as long as there exists a minimum 3' overhang of 2 nt to initiate degradation. We identified a Snp mutant and used it to test whether Snp plays a role in regulating histone mRNA degradation or RNAi in vivo. Snp mutant flies are viable, and display no obvious developmental abnormalities. The expression pattern and level of histone H3 mRNA in Snp mutant embryos and third instar imaginal eye discs was indistinguishable from wild type, suggesting that Snp does not play a significant role in the turnover of histone mRNA at the end of the S phase. The loss of Snp was also unable to enhance the silencing capability of two different RNAi transgenes targeting the white and yellow genes, suggesting that Snp does not negatively modulate RNAi. Therefore, Snp is a nonessential exonuclease that is not a functional ortholog of either 3'hExo or ERI-1.     

CRN-1, a Caenorhabditis elegans FEN-1 homologue,cooperates with CPS-6/EndoG to promote apoptotic DNA degradation

Oligonucleosomal fragmentation of chromosomes in dying cells is a hallmark of apoptosis. Little is known about how it is executed or what cellular components are involved. We show that crn-1, a Caenorhabditis elegans homologue of human flap endonuclease-1 (FEN-1) that is normally involved in DNA replication and repair, is also important for apoptosis. Reduction of crn-1 activity by RNA interference resulted in cell death phenotypes similar to those displayed by a mutant lacking the mitochondrial endonuclease CPS-6/endonuclease G. CRN-1 localizes to nuclei and can associate and cooperate with CPS-6 to promote stepwise DNA fragmentation, utilizing the endonuclease activity of CPS-6 and both the 5'-3' exonuclease activity and a previously uncharacterized gap-dependent endonuclease activity of CRN-1. Our results suggest that CRN-1/FEN-1 may play a critical role in switching the state of cells from DNA replication/repair to DNA degradation during apoptosis.     

ARE-mRNA degradation requires the 5'-3' decay pathway

As an important mode of suppressing gene expression, messenger RNAs containing an AU-rich element (ARE) in the 3' untranslated region are rapidly degraded in the cytoplasm. ARE-mediated mRNA decay (AMD) is initiated by deadenylation, and in vitro studies have indicated that subsequent degradation occurs in the 3'-5' direction through a complex of exonucleases termed the exosome. An alternative pathway of mRNA degradation occurs at processing bodies, cytoplasmic foci that contain decapping enzymes, the 5'-3' exonuclease Xrn1 and the Lsm1-7 heptamer. To determine which of the two pathways is important for AMD in live cells, we targeted components of both pathways using short interfering RNA in human HT1080 cells. We show that Xrn1 and Lsm1 are essential for AMD. On the other side, out of three exosome components tested, only knockdown of PmScl-75 caused a strong inhibition of AMD. Our results show that mammalian cells, similar to yeast, require the 5'-3' Xrn1 pathway to degrade ARE-mRNAs.     

Plant mitochondrial polyadenylated mRNAs are degraded by a 3'- to 5'-exoribonuclease activity, which proceeds unimpeded by stable secondary structures

Recently, we and others have reported that mRNAs may be polyadenylated in plant mitochondria, and that polyadenylation accelerates the degradation rate of mRNAs. To further characterize the molecular mechanisms involved in plant mitochondrial mRNA degradation, we have analyzed the polyadenylation and degradation processes of potato atp9 mRNAs. The overall majority of polyadenylation sites of potato atp9 mRNAs is located at or in the vicinity of their mature 3'-extremities. We show that a 3'- to 5'-exoribonuclease activity is responsible for the preferential degradation of polyadenylated mRNAs as compared with non-polyadenylated mRNAs, and that 20-30 adenosine residues constitute the optimal poly(A) tail size for inducing degradation of RNA substrates in vitro. The addition of as few as seven non-adenosine nucleotides 3' to the poly(A) tail is sufficient to almost completely inhibit the in vitro degradation of the RNA substrate. Interestingly, the exoribonuclease activity proceeds unimpeded by stable secondary structures present in RNA substrates. From these results, we propose that in plant mitochondria, poly(A) tails added at the 3' ends of mRNAs promote an efficient 3'- to 5'- degradation process.     

Polyadenylation accelerates degradation of chloroplast mRNA.

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.     

Inhibition of 5' to 3' mRNA degradation under stress conditions in Saccharomyces cerevisiae: from GCN4 to MET16

After deadenylation, most cytoplasmic mRNAs are decapped and digested by 5' to 3' exonucleases in Saccharomyces cerevisiae. Capped and deadenylated mRNAs are degraded to a lesser extent by 3' to 5' exonucleases. We have used a method, based on the electroporation of in vitro synthetised mRNAs, to study the relative importance of these two exonucleolytic pathways under stress conditions. We show that derepression of GCN4 upon amino acid starvation specifically limits the 5'-to-3'-degradation pathway. Because adenosine 3'-5' biphosphate (pAp), which is produced by Met16p, inhibits this degradation pathway to a comparable extent, we were prompted to analyse the role of Met16p in this phenomenon. We show that the inhibitory effects of amino acid limitation on 5' to 3' mRNA degradation are absent in a met16 mutant. We therefore conclude that the GCN4 dependence of MET16 expression is responsible for the decrease in 5' to 3' digestion under stress conditions and that cells use pAp as a signal to limit 5' to 3' RNA degradation under stress conditions. Because 3' to 5' mRNA degradation is unaffected, the relative importance of this pathway in the decay of certain RNAs may be increased under stress conditions.     

A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.     

Crystallographic structure of the nuclease domain of 3'hExo, a DEDDh family member, bound to rAMP

A human 3'-5'-exoribonuclease (3'hExo) has recently been identified and shown to be responsible for histone mRNA degradation. Functionally, 3'hExo and a stem-loop binding protein (SLBP) target opposite faces of a unique highly conserved stem-loop RNA scaffold towards the 3' end of histone mRNA, which is composed of a 6 bp stem and a 4 nt loop, followed by an ACCCA sequence. Its Caenorhabditis elegans homologue, ERI-1, has been shown to degrade small interfering RNA in vitro and to function as a negative regulator of RNA interference in neuronal cells. We have determined the structure of the nuclease domain (Nuc) of 3'hExo complexed with rAMP in the presence of Mg2+ at 1.6 A resolution. The Nuc domain adopts an alpha/beta globular fold, with four acidic residues coordinating a binuclear metal cluster within the active site, whose topology is related to DEDDh exonuclease family members, despite a very low level of primary sequence identity. The two magnesium cations in the Nuc active site are coordinated to D134, E136, D234 and D298, and together with H293, which can potentially act as a general base, provide a platform for hydrolytic cleavage of bound RNA in the 3' --> 5' direction. The bound rAMP is positioned within a deep active-site pocket, with its purine ring close-packed with the hydrophobic F185 and L189 side-chains and its sugar 2'-OH and 3'-OH groups hydrogen bonded to backbone atoms of Nuc. There are striking similarities between the active sites of Nuc and epsilon186, an Escherichia coli DNA polymerase III proofreading domain, providing a common hydrolytic cleavage mechanism for RNA degradation and DNA editing, respectively.     

The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization

RNase II is a 3'-5' exoribonuclease that processively hydrolyzes single-stranded RNA generating 5' mononucleotides. This enzyme contains a catalytic core that is surrounded by three RNA-binding domains. At its C terminus, there is a typical S1 domain that has been shown to be critical for RNA binding. The S1 domain is also present in the other major 3'-5' exoribonucleases from Escherichia coli: RNase R and polynucleotide phosphorylase (PNPase). In this report, we examined the involvement of the S1 domain in the different abilities of these three enzymes to overcome RNA secondary structures during degradation. Hybrid proteins were constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability were examined. The results revealed that both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. Moreover, the S1 domains investigated are not equivalent. Furthermore, we demonstrate that S1 is neither responsible for the ability to overcome secondary structures during RNA degradation, nor is it related to the size of the final product generated by each enzyme. In addition, we show that the S1 domain from PNPase is able to induce the trimerization of the RNaseII-PNP hybrid protein, indicating that this domain can have a role in the biogenesis of multimers.     

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.     

Rat1p and Rai1p function with the nuclear exosome in the processing and degradation of rRNA precursors

Exoribonucleases function in the processing and degradation of a variety of RNAs in all organisms. These enzymes play a particularly important role in the maturation of rRNAs and in a quality-control pathway that degrades rRNA precursors upon inhibition of ribosome biogenesis. Strains with defects in 3'-5' exoribonucleolytic components of the RNA processing exosome accumulate polyadenylated precursor rRNAs that also arise in strains with ribosome biogenesis defects. These findings suggested that polyadenylation might target pre-rRNAs for degradation by the exosome. Here we report experiments that indicate a role for the 5'-3' exoribonuclease Rat1p and its associated protein Rai1p in the degradation of poly(A)(+) pre-rRNAs. Depletion of Rat1p enhances the amount of poly(A)(+) pre-rRNA that accumulates in strains deleted for the exosome subunit Rrp6p and decreases their 5' heterogeneity. Deletion of RAI1 results in the accumulation of poly(A)(+) pre-rRNAs, and inhibits Rat1p-dependent 5'-end processing and Rrp6p-dependent 3'-end processing of 5.8S rRNA. RAT1 and RAI1 mutations cause synergistic growth defects in the presence of rrp6-Delta, consistent with the interdependence of 5'-end and 3'-end processing pathways. These findings suggest that Rai1p may coordinate the 5'-end and 3'-end processing and degradation activities of Rat1p and the nuclear exosome.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro.

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.