Cytoplasmic mRNA Degradation Research Articles

The Ccr4-Not Complex Interacts with the mRNA Export Machinery

BackgroundThe Ccr4-Not complex is a key eukaryotic regulator of gene transcription and cytoplasmic mRNA degradation. Whether this complex also affects aspects of post-transcriptional gene regulation, such as mRNA export, remains largely unexplored. Human Caf1 (hCaf1), a Ccr4-Not complex member, interacts with and regulates the arginine methyltransferase PRMT1, whose targets include RNA binding proteins involved in mRNA export. However, the functional significance of this regulation is poorly understood.Methodology/Principal FindingsHere we demonstrate using co-immunoprecipitation approaches that Ccr4-Not subunits interact with Hmt1, the budding yeast ortholog of PRMT1. Furthermore, using genetic and biochemical approaches, we demonstrate that Ccr4-Not physically and functionally interacts with the heterogenous nuclear ribonucleoproteins (hnRNPs) Nab2 and Hrp1, and that the physical association depends on Hmt1 methyltransferase activity. Using mass spectrometry, co-immunoprecipitation and genetic approaches, we also uncover physical and functional interactions between Ccr4-Not subunits and components of the nuclear pore complex (NPC) and we provide evidence that these interactions impact mRNA export.Conclusions/SignificanceTaken together, our findings suggest that Ccr4-Not has previously unrealized functional connections to the mRNA processing/export pathway that are likely important for its role in gene expression. These results shed further insight into the biological functions of Ccr4-Not and suggest that this complex is involved in all aspects of mRNA biogenesis, from the regulation of transcription to mRNA export and turnover.

The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4–Not complex contribute to the prevention of cell death and senescence

A key step in cytoplasmic mRNA degradation is the shortening of the poly(A) tail, which involves several deadenylase enzymes. Relatively little is known about the importance of these enzymes for the cellular physiology. Here we focused on the role of the highly similar Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the Ccr4-Not complex. In addition to a role in cell proliferation, Ccr4a and Ccr4b play a role in cell survival, in contrast to the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits or the CNOT1 and CNOT3 noncatalytic subunits of the Ccr4-Not complex. Underscoring the differential contributions of the deadenylase subunits, we found that knockdown of Caf1a/Caf1b or Ccr4a/Ccr4b differentially affects the formation of cytoplasmic foci by processing-body components. Furthermore, we demonstrated that the amino-terminal leucine-rich repeat (LRR) domain of Ccr4b influenced its subcellular localization but was not required for the deadenylase activity of Ccr4b. Moreover, overexpression of Ccr4b lacking the LRR domain interfered with cell cycle progression but not with cell viability. Finally, gene expression profiling indicated that distinct gene sets are regulated by Caf1a/Caf1b and Ccr4a/Ccr4b and identified Ccr4a/Ccr4b as a key regulator of insulin-like growth factor-binding protein 5, which mediates cell cycle arrest and senescence via a p53-dependent pathway.

LSM1 over-expression in Saccharomyces cerevisiae depletes U6 snRNA levels

Lsm1 is a component of the Lsm1-7 complex involved in cytoplasmic mRNA degradation. Lsm1 is over-expressed in multiple tumor types, including over 80% of pancreatic tumors, and increased levels of Lsm1 protein have been shown to induce carcinogenic effects. Therefore, understanding the perturbations in cell process due to increased Lsm1 protein may help to identify possible therapeutics targeting tumors over-expressing Lsm1. Herein, we show that LSM1 over-expression in the yeast Saccharomyces cerevisiae inhibits growth primarily due to U6 snRNA depletion, thereby altering pre-mRNA splicing. The decrease in U6 snRNA levels causes yeast strains over-expressing Lsm1 to be hypersensitive to loss of other proteins required for production or function of the U6 snRNA, supporting a model wherein excess Lsm1 reduces the availability of the Lsm2-7 proteins, which also assemble with Lsm8 to form a complex that binds and stabilizes the U6 snRNA. Yeast strains over-expressing Lsm1 also display minor alterations in mRNA decay and demonstrate increased susceptibility to mutations inhibiting cytoplasmic deadenylation, a process required for both 5′-to-3′ and 3′-to-5′ pathways of exonucleolytic decay. These results suggest that inhibition of splicing and/or deadenylation may be effective therapies for Lsm1-over-expressing tumors.

Crystal structure of an archaeal Ski2p‐like protein from Pyrococcus horikoshii OT3

The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3' to 5' cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H-box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p-like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA-dependent ATPase activity with an optimal temperature at approximately 90 degrees C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 A using single-wavelength anomalous dispersion (SAD) and selenomethionyl (Se-Met)-substituted protein. Ph1280p comprises four subdomains; the two N-terminal subdomains (N1 and N2) fold into an RecA-like architecture with the conserved helicase motifs, while the two C-terminal subdomains (C1 and C2) fold into alpha-helical structures containing a winged helix (WH)-fold and helix-hairpin-helix (HhH)-fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C-terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2-like helicase and would play a critical role in the 3' to 5' cytoplasmic mRNA degradation in the Ski complex.

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.

RNA Gel Shift Assays for Analysis of Hormone Control of mRNA Stability

Publisher Summary This chapter focuses on the regulation of the cytoplasmic stability of specific mRNAs. The level of an mRNA in the cytoplasm represents a balance between the rate at which the mRNA precursor is synthesized in the nucleus and the rates of nuclear RNA processing, and export and cytoplasmic mRNA degradation. While it has long been known that steroid hormones regulate mRNA stability, until the past few years progress in this area was limited by the dearth of information on the general pathways of eukaryotic mRNA degradation, the lack of suitable cell culture models, and the absence of appropriate tools for analyzing mRNA stability and the protein–nucleic acid interactions that control this process. Studies of the hormone regulation of mRNA degradation employ an evolving constellation of technologies. The chapter focuses on the use of RNA gel mobility shift assays to monitor the levels of hormone-regulated mRNA-binding proteins and to study their interactions with specific mRNAs. Hormones regulate the levels of a number of mRNA-binding proteins, and thereby modulate mRNA stability. RNA gel shift assays show that polysome salt extracts from liver cells of estrogen-treated Xenopus exhibit increased binding of vigilin to a crucial segment of the vitellogenin mRNA 3′-untranslated region. The basic principle of DNA and RNA gel mobility shift assays is that polyacrylamide gel electrophoresis is used to separate a labeled nucleic acid probe from the protein–nucleic acid complex.

Regulation of pathways of mRNA destabilization and stabilization

The level of an mRNA in the cytoplasm represents a balance between the rate at which the mRNA precursor is synthesized in the nucleus and the rates of nuclear RNA processing and export and cytoplasmic mRNA degradation. Although most studies of gene expression have focused on gene transcription and in the area of eukaryotic mRNA degradation, but to provide a short general discussion of the importance of mRNA degradation and its regulation and a brief overview of recent findings and present knowledge. The overview is followed by a more in-depth discussion of one of the several pathways for mRNA degradation. We concentrate on the pathway for regulated mRNA degradation mediated by mRNA-binding proteins and endonucleases that cleave within the body of mRNAs. As a potential example of this type of control, we focus on the regulated degradation of the egg yolk precursor protein vitellogenin on the mRNA-binding protein vigilin and the mRNA endonuclease polysomal ribonuclease 1 (PMR-1).

Novel features of the XRN-family in Arabidopsis : Evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm

The 5'-3' exoribonucleases Xrn1p and Xrn2p/Rat1p function in the degradation and processing of several classes of RNA in Saccharomyces cerevisiae. Xrn1p is the main enzyme catalyzing cytoplasmic mRNA degradation in multiple decay pathways, whereas Xrn2p/Rat1p functions in the processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Much less is known about the XRN-like proteins of multicellular eukaryotes; however, differences in their activities could explain differences in mRNA degradation between multicellular and unicellular eukaryotes. One such difference is the lack in plants and animals of mRNA decay intermediates like those generated in yeast when Xrn1p is blocked by poly(G) tracts that are inserted within mRNAs. We investigated the XRN-family in Arabidopsis thaliana and found it to have several novel features. First, the Arabidopsis genome contains three XRN-like genes (AtXRNs) that are structurally similar to Xrn2p/Rat1p, a characteristic unique to plants. Furthermore, our experimental results and sequence database searches indicate that Xrn1p orthologs may be absent from higher plants. Second, the lack of poly(G) mRNA decay intermediates in plants cannot be explained by the activity of the AtXRNs, because they are blocked by poly(G) tracts. Finally, complementation of yeast mutants and localization studies indicate that two of the AtXRNs likely function in the nucleus, whereas the third acts in the cytoplasm. Thus, the XRN-family in plants is more complex than in other eukaryotes, and, if an XRN-like enzyme plays a role in mRNA decay in plants, the likely participant is a cytoplasmic Xrn2p/Rat1p ortholog, rather than an Xrn1p ortholog.

Mutational analysis of exoribonuclease I from Saccharomyces cerevisiae.

Exoribonuclease I from yeast is a 175 kDa protein that is responsible for the majority of cytoplasmic mRNA degradation. Alignment of the Xrn1p sequence with homologs from yeast as well as from higher eukaryotes suggests that the protein is composed of several domains: two acidic N-terminal domains which likely contain the exonuclease, a basic middle domainand a basic C-terminal domain. Deletion analysisdemonstrated that the C-terminus is dispensable for most in vivo and in vitro functions but confers a dominant negative growth inhibition when expressed at high levels. This growth inhibition is not due to the exonuclease function of the protein. To identify specific residues responsible for in vivo function, a screen was carried out for non-complementing missense mutations. Fourteen single point mutations were identified that altered highly conserved amino acids within the first N-terminal domain of Xrn1p. All of the mutations reduced exonuclease activity measured in vivo and in vitro using affinity-purified proteins. The mutants fell into two phenotypic classes, those that reduced or abolished exonuclease activity without qualitatively changing the products of RNA degradation and those that gave rise to novel degradation intermediates on certain RNAs.

Thyroid hormone regulation of malic enzyme synthesis. Dual tissue-specific control.

The regulatory mechanism(s) involved in the tissue-specific induction of cytosolic malic enzyme (EC 1.1.1.40) by triiodothyronine (T3) have been investigated in rat liver and heart. In these two tissues, cellular malic enzyme mRNA accumulates to different extents in response to hormonal stimulation (11-16- and 3-4-fold above the respective basal levels) (Dozin, B., Magnuson, M. A., and Nikodem, V. M. (1985) Biochemistry 24, 5581-5586). To gain further insight into this pretranslational control, nuclear in vitro run-off transcription assays were performed and correlated with the levels of malic enzyme RNA sequences in cytoplasm. The data demonstrate that the rate of transcription of the malic enzyme gene is stimulated by T3 to similar extents in liver and heart (3-4-fold above the basal activity). In liver, T3 also promotes an additional increase in cellular malic enzyme mRNA. This additional effect could be due to a tissue-specific change in the rate of degradation of cytoplasmic mRNA or to an effect on malic enzyme mRNA in the nucleus.

Tissue-specific control of rat malic enzyme activity and messenger RNA levels by a high carbohydrate diet.

In euthyroid rats fed a high carbohydrate fat-free diet for 10 days, the mass of cellular malic enzyme mRNA in liver is increased 7- to 8-fold above the basal level. Malic enzyme activity is stimulated to the same extent. This effect does not result from an increase either in the transcriptional activity of the malic enzyme gene, as determined by nuclear run-off transcription assay, or in the content of intranuclear malic enzyme RNA sequences. Mathematical modeling shows that this increase in cytoplasmic mRNA is compatible with retarded degradation of cytoplasmic mRNA. Regulation of malic enzyme by carbohydrates is liver-specific, since no response is observed in the following nonhepatic tissues: brain, heart, spleen, kidney, testis, and lung. Furthermore, the amplitude of the response in liver depends on the thyroid state of the animals, being lower (by a factor of approximately 4) in hypothyroidism and higher (12- to 15-fold) when normal animals are injected simultaneously with a daily dose of 15 micrograms of triiodothyronine per 100 g of body weight for 10 days. Since thyroid hormones regulate liver malic enzyme synthesis predominantly at the nuclear level and carbohydrates at the cytoplasmic level, the additive effect of triiodothyronine and a high carbohydrate diet on the activity of malic enzyme is readily explicable.

Fate of histone messenger RNA in synchronized HeLa cells in the absence of initiation of protein synthesis.

The fate of cytoplasmic histone mRNA was studied under conditions in which initiation of protein synthesis in synchronized HeLa cells is S phase was blocked by increasing the osmolarity of the growth medium with NaCl. In contrast to the interruption of DNA replication with hydroxyurea, which results in an exponential degradation of translatable histone mRNA with a half-life of about 10-13 min, blocking the initiation of protein synthesis leads to only a marginal loss of biologically active histone mRNA in the cytoplasm. When the initiation of protein synthesis was interrupted by treating cells with 150 mM NaCl, 40-50% of the total cytoplasmic histone mRNA previously translated in polyribosomes appears in the cytoplasm integrated into mRNA-protein particle(s) sedimenting between 15 S and 30 S. On the other hand, in untreated S-phase cells or in cells blocked with hydroxyurea only 3-6% of the total translatable histone mRNA is found in the cytoplasm not bound to ribosomes or their subunits. In addition, the degradation of histone mRNA in hydroxyurea-blocked S-phase cells is prevented when the initiation of protein synthesis is inhibited with NaCl. These studies clearly indicate that the inhibition of initiation of protein synthesis per se is not the cause for the rapid degradation of cytoplasmic histone mRNA observed when DNA replication is turned off and that the inactivation of these mRNAs is a process dependent on continuous protein synthesis.