Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.     

Analysis of the products of mRNA decapping and 3'-to-5' decay by denaturing gel electrophoresis

The majority of mRNA turnover is mediated either by mRNA decapping/5'-to-3' decay or exosome-mediated 3'-to-5' exonucleolytic decay. Current assays to assess mRNA decapping in vitro using cap-labeled RNA substrates rely on one-dimensional thin layer chromatography. This approach does not, however, resolve free phosphate from 7meGDP, the product of Dcp1p-mediated mRNA decapping. This can result in misinterpretation of the levels of mRNA decapping due to the generation of free phosphate following the action of the unrelated scavenger decapping activity on the products of exosome-mediated decay. In this report, we describe a simple denaturing acrylamide gel-based assay that faithfully resolves all of the possible products that can be generated from cap-labeled RNA substrates by turnover enzymes present in cell extracts. This approach allows a one-step assay to quantitatively assess the contributions of the exosome and DCP-1-type decapping on turnover of an RNA substrate in vitro. We have applied this assay to recalculate the effect of competition of cap-binding proteins on decapping in yeast. In addition, we have used the assay to confirm observations made on regulated mRNA decapping in mammalian extracts that contain much higher levels of exosome activity than yeast extracts.     

Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping

The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.     

Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p

The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3'-5' cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3'-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with coimmunoprecipitation experiments. We also test the functional significance of these interactions in vivo. Our results suggest that within the Ski complex, Ski3p serves as a scaffold protein with its C terminus interacting with Ski8p, and the sub-C terminus interacting with Ski2p, while no direct interaction between Ski2p and Ski8p was found. Ski7p interacts with the Ski complex via its interaction with Ski8p and Ski3p. In addition, inactivating the Ski complex by mutating conserved residues in the DEVH helicase motif of Ski2 did not abrogate its interaction with Ski7p, indicating that Ski2p function is not necessary for this interaction.     

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo

The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.     

Identification of an RNase J ortholog in Sulfolobus solfataricus: implications for 5'-to-3' directional decay and 5'-end protection of mRNA in Crenarchaeota

In both Bacteria and Eukaryotes, degradation is known to start at the 5' and at the 3' extremities of mRNAs. Until the recent discovery of 5'-to-3' exoribonucleases in hyperthermophilic Euryarchaeota, the exosome was assumed to be the key enzyme in mRNA degradation in Archaea. By means of zymogram assays and bioinformatics, we have identified a 5'-to-3' exoribonuclease activity in the crenarchaeum Sulfolobus solfataricus (Sso), which is affected by the phosphorylation state of the 5'-end of the mRNA. The protein comprises typical signature motifs of the β-CASP family of metallo-β-lactamases and was termed Sso-RNAse J. Thus, our study provides the first evidence for a 5'-to-3' directional mRNA decay pathway in the crenarchaeal clade of Archaea. In Bacteria the 5'-end of mRNAs is often protected by a tri-phosphorylated 5'-terminus and/or by stem-loop structures, while in Eukaryotes the cap-binding complex is responsible for this task. Here, we show that binding of translation initiation factor a/eIF2(γ) to the 5'-end of mRNA counteracts the 5'-to-3' exoribonucleolytic activity of Sso-RNase J in vitro. Hence, 5'-to-3' directional decay and 5'-end protection appear to be conserved features of mRNA turnover in all kingdoms of life.     

A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p

Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)(+) mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors, rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)(+) mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3'-5' exonuclease core domain within Rrp6p. As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3'-5' exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)(+) mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.     

5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA

Although the primary mechanism of eukaryotic messenger RNA decay is exoribonucleolytic degradation in the 5'-to-3' orientation, it has been widely accepted that Bacteria can only degrade RNAs with the opposite polarity, i.e. 3' to 5'. Here we show that maturation of the 5' side of Bacillus subtilis 16S ribosomal RNA occurs via a 5'-to-3' exonucleolytic pathway, catalyzed by the widely distributed essential ribonuclease RNase J1. The presence of a 5'-to-3' exoribonuclease activity in B. subtilis suggested an explanation for the phenomenon whereby mRNAs in this organism are stabilized for great distances downstream of "roadblocks" such as stalled ribosomes or stable secondary structures, whereas upstream sequences are never detected. We show that a 30S ribosomal subunit bound to a Shine Dalgarno-like element (Stab-SD) in the cryIIIA mRNA blocks exonucleolytic progression of RNase J1, accounting for the stabilizing effect of this element in vivo.     

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.     

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3' untranslated regions (3' UTRs) of target mRNAs. In yeast, Puf3p binds the 3' UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3' UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3' UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3' UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain.     

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.     

Different regulation of the p53 core domain activities 3'-to-5' exonuclease and sequence-specific DNA binding

In this study we further characterized the 3'-5' exonuclease activity intrinsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-terminal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activity by at least 10-fold, indicating that this activity, like sequence-specific DNA binding, is negatively regulated by the C-terminal basic regulatory domain of p53. However, treatments which activated sequence-specific DNA binding of p53, like binding of the monoclonal antibody PAb421, which recognizes a C-terminal epitope on p53, or a higher phosphorylation status, strongly inhibited the p53 exonuclease activity. This suggests that at least on full-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Following up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombination intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such substrates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to the binding assay indeed started the p53 exonuclease and promoted rapid degradation of the bound, but not of the unbound, substrate, indicating that specifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.