A role for the exosome in the in vivo degradation of unstable mRNAs

In mammals, the mRNAs encoding many proteins involved in inflammation bear destabilizing AU-rich elements (AREs) in the 3'-untranslated region. The exosome, a complex of 3' --> 5' exonucleases, is rate limiting in the destruction of such mRNAs in a mammalian in vitro system, but a role in vivo has not been demonstrated. The phenomenon of ARE-mediated degradation also occurs in the protist parasite Trypanosoma brucei. Messenger RNAs with 3'-untranslated region U-rich elements, which strongly resemble AREs, are extremely unstable in the trypanosome form that parasitizes mammals. The first step in degradation of these mRNAs in vivo is rapid destruction of the 3'-untranslated region; subsequently the mRNA is destroyed by exonucleases acting in both 5' --> 3' and 3' --> 5' directions. We here investigated the roles of three subunits of the trypanosome exosome complex, RRP45, RRP4, and CSL4, in this process, depleting the individual subunits in vivo by inducible RNA interference. RRP45 depletion, which probably disrupts exosome integrity, caused a delay in the onset of degradation of the very unstable RNAs, but did not affect degradation of more stable species. Depletion of RRP4 or CSL4 does not affect the stability of the residual exosome and did not change mRNA degradation kinetics. We conclude that the exosome is required for the initiation of rapid degradation of unstable mRNAs in trypanosomes.     

A role for Caf1 in mRNA deadenylation and decay in trypanosomes and human cells

The eukaryotic Ccr4/Caf1/Not complex is involved in deadenylation of mRNAs. The Caf1 and Ccr4 subunits both potentially have deadenylating enzyme activity. We investigate here the roles of Ccr4 and Caf1 in deadenylation in two organisms that separated early in eukaryotic evolution: humans and trypanosomes. In Trypanosoma brucei, we found a complex containing CAF1, NOT1, NOT2 and NOT5, DHH1 and a possible homologue of Caf130; no homologue of Ccr4 was found. Trypanosome CAF1 has deadenylation activity, and is essential for cell survival. Depletion of trypanosome CAF1 delayed deadenylation and degradation of constitutively expressed mRNAs. Human cells have two isozymes of Caf1: simultaneous depletion of both inhibited degradation of an unstable reporter mRNA. In both species, depletion of Caf1 homologues inhibited deadenylation of bulk RNA and resulted in an increase in average poly(A) tail length.     

Inhibition of mRNA deadenylation and degradation by different types of cell stress

We have previously observed rapid and strong inhibition of mRNA deadenylation and degradation in response to UV-B light [Gowrishankar et al., Biol. Chem. 386 (2005), pp. 1287-1293]. Expression analysis using a microarray for inflammatory genes showed that UV-B light induces stabilization of all short-lived mRNAs assayed. Stabilization was observed in HeLa cells, as well as in the keratinocyte line HaCaT. It affected constitutively expressed mRNA species, as well as species induced by the inflammatory cytokine IL-1. Many of the latter encode proteins involved in inflammation, suggesting that stress-induced inhibition of mRNA deadenylation contributes to changes in inflammatory gene expression. Deadenylation and degradation of tet-off-expressed mRNAs were also inhibited upon exposure to H2O2. However, scavengers of reactive oxygen species did not interfere with UV-B-induced inhibition of degradation, arguing against the involvement of UV-induced H2O2 in these effects of UV-B light. Heat shock and hyperosmolarity also inhibited mRNA deadenylation and degradation, whereas gamma-radiation did not. Thus, inhibition of mRNA deadenylation and degradation is a cellular response elicited by several but not all inducers of cell stress.     

SsrA-mediated trans-translation plays a role in mRNA quality control by facilitating degradation of truncated mRNAs

An important unsolved question regarding the bacterial SsrA system is the fate of target mRNAs replaced by SsrA RNA during trans-translation. The aim of the present study is to address the potential role of SsrA system in mRNA quality control, focusing on truncated mRNAs that are expected to arise from 3'-to-5' exonucleolytic attack. We found that significant amounts of truncated mRNAs and polypeptides were produced from genes lacking a rho-independent terminator in SsrA-deficient cells. These truncated mRNAs, hence truncated polypeptides, were no longer observed in the presence of SsrA RNA. The data indicate that the SsrA system facilitates degradation of "nonstop" mRNAs by presumably removing the stalled ribosomes. Furthermore, analysis of affinity-purified proteins indicated that truncated polypeptides could be produced even from a gene with an intact rho-independent terminator, although less efficiently, implying that C-terminally truncated proteins and 3'-truncated mRNA may be produced from virtually all protein-coding genes. We conclude that the SsrA system not only promotes the degradation of incomplete polypeptides but also minimizes the synthesis of incomplete polypeptides by facilitating the degradation of truncated mRNAs that are produced in cells.     

The role of the AU-rich elements of mRNAs in controlling translation

Adenosine- and uridine-rich elements (AREs) located in 3'-untranslated regions are the best-known determinants of RNA instability. These elements have also been shown to control translation in certain mRNAs, including mRNAs for prominent pro-inflammatory and tumor growth-related proteins, and physiological anti-inflammatory processes that target ARE-controlled translation of mRNAs coding for pro-inflammatory proteins have been described. A major research effort is now being made to understand the mechanisms by which the translation of these mRNAs is controlled and the signalling pathways involved. This review focuses on the role of ARE-containing gene translation in inflammation, and the disease models that have improved our understanding of ARE-mediated translational control.     

The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G₁ fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G₁ fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOT complex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.     

Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes

The surface of Trypanosoma brucei is dominated by glycosyl-phosphatidylinositol (GPI)-anchored proteins, and endocytosis is clathrin dependent. The vast majority of internalized GPI-anchored protein is efficiently recycled, while the processes by which transmembrane domain (TMD) proteins are internalized and sorted are unknown. We demonstrate that internalization of invariant surface glycoprotein (ISG)65, a trypanosome TMD protein, involves ubiquitylation and also requires clathrin. We find a hierarchical requirement for cytoplasmic lysine residues in internalization and turnover, and a single position-specific lysine is sufficient for degradation, surface removal and attachment of oligoubiquitin chains. Ubiquitylation is context dependent as provision of additional lysine residues by C-terminal fusion of neuronal precursor cell-expressed developmentally downregulated protein (NEDD)8 fails to support ubiquitylation. Attachment of NEDD8 leads to degradation by a second ubiquitin-independent pathway. Moreover, degradation of ubiquitylated or NEDDylated substrate takes place in an acidic compartment and is proteosome independent. Significantly, in non-opisthokont lineages, Rsp5p or c-Cbl, the E3 ubiquitin ligases acting on endocytic cargo, are absent but Uba1 class genes are present and are required for cell viability and ISG65 ubiquitylation. Hence, ubiquitylation is an evolutionarily conserved mechanism for internalization of surface proteins, but aspects of the machinery differ substantially between the major eukaryotic lineages.     

The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes

RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.     

The role of two superoxide dismutase mRNAs in rye aluminium tolerance

Aluminium (Al) is the main factor that limits crop production in acidic soils. There is evidence that antioxidant enzymes such as superoxide dismutase (SOD) play a key role against Al‐induced oxidative stress in several plant species. Rye is one of the most Al‐tolerant cereals and exudes both citrate and malate from the roots in response to Al. The role of SOD against Al‐induced oxidative stress has not been studied in rye. Al accumulation, lipid peroxidation, H₂O₂ production and cell death were significantly higher in sensitive than in tolerant rye cultivars. Also, we characterised two genes for rye SOD: ScCu/ZnSOD and ScMnSOD. These genes were located on the chromosome arms of 2RS and 3RL, respectively, and their corresponding hypothetical proteins were putatively classified as cytosolic and mitochondrial, respectively. The phylogenetic relationships indicate that the two rye genes are orthologous to the corresponding genes of other Poaceae species. In addition, we studied Al‐induced changes in the expression profiles of mRNAs from ScCu/ZnSOD and ScMnSOD in the roots and leaves of tolerant Petkus and sensitive Riodeva rye. These genes are mainly expressed in roots in both ryes, their repression being induced by Al. The tolerant cultivar has more of both mRNAs than the sensitive line, indicating that they are probably involved in Al tolerance.     

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.