An RNA granule for translation quality control in Saccharomyces cerevisiae.

Cytoplasmic RNA granules compartmentalize phases of the translation cycle in eukaryotes. We previously reported the localization of oxidized RNA to cytoplasmic foci called oxidized RNA bodies (ORBs) in human cells. We show here that ORBs are RNA granules in Saccharomyces cerevisiae. Several lines of evidence support a role for ORBs in the compartmentalization of no-go decay and ribosome quality control, the translation quality control pathways that recognize and clear aberrant mRNAs, including those with oxidized bases. Translation is required by these pathways and ORBs. Translation quality control factors localize to ORBs. A substrate of translation quality control, a stalled mRNA-ribosome-nascent-chain complex, localizes to ORBs. Translation quality control mutants have altered ORB numbers, sizes or both. In addition, we identify 68 ORB proteins by immunofluorescence staining directed by proteomics, which further support their role in translation quality control and reveal candidate new factors for these pathways.

Localized control of oxidized RNA.

The oxidation of biological molecules by reactive oxygen species (ROS) can render them inactive or toxic. This includes the oxidation of RNA, which appears to underlie the detrimental effects of oxidative stress, aging and certain neurodegenerative diseases. Here, we investigate the management of oxidized RNA in the chloroplast of the green alga Chlamydomonas reinhardtii. Our immunofluorescence microscopy results reveal that oxidized RNA (with 8-hydroxyguanine) is localized in the pyrenoid, a chloroplast microcompartment where CO2 is assimilated by the Calvin cycle enzyme Rubisco. Results of genetic analyses support a requirement for the Rubisco large subunit (RBCL), but not Rubisco, in the management of oxidized RNA. An RBCL pool that can carry out such a 'moonlighting' function is revealed by results of biochemical fractionation experiments. We also show that human (HeLa) cells localize oxidized RNA to cytoplasmic foci that are distinct from stress granules, processing bodies and mitochondria. Our results suggest that the compartmentalization of oxidized RNA management is a general phenomenon and therefore has some fundamental significance.

Each Cellular Compartment Has a Characteristic Protein Reactive Cysteine Ratio Determining Its Sensitivity to Oxidation.

Signaling and detoxification of Reactive Oxygen Species (ROS) are important patho-physiologcal processes. Despite this, we lack comprehensive information on individual cells and cellular structures and functions affected by ROS, which is essential to build quantitative models of the effects of ROS. The thiol groups from cysteines (Cys) in proteins play a major role in redox defense, signaling, and protein function. In this study, we show that the proteins in each subcellular compartment contain a characteristic Cys amount. Using a fluorescent assay for -SH in thiolate form and amino groups in proteins, we show that the thiolate content correlates with ROS sensitivity and signaling properties of each compartment. The highest absolute thiolate concentration was found in the nucleolus, followed by the nucleoplasm and cytoplasm whereas protein thiolate groups per protein showed an inverse pattern. In the nucleoplasm, protein reactive thiols concentrated in SC35 speckles, SMN, and the IBODY that accumulated oxidized RNA. Our findings have important functional consequences, and explain differential sensitivity to ROS.

Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1.

Understanding how oxidatively damaged RNA is handled intracellularly is of relevance due to the link between oxidized RNA and the progression/development of some diseases as well as aging. Among the ribonucleases responsible for the decay of modified (chemically or naturally) RNA is the exonuclease Xrn-1, a processive enzyme that catalyzes the hydrolysis of 5'-phosphorylated RNA in a 5'→3' direction. We set out to explore the reactivity of this exonuclease towards oligonucleotides (ONs, 20-nt to 30-nt long) of RNA containing 8-oxo-7,8-dihydroguanosine (8-oxoG), obtained via solid-phase synthesis. The results show that Xrn-1 stalled at sites containing 8-oxoG, evidenced by the presence of a slower moving band (via electrophoretic analyses) than that observed for the canonical analogue. The observed fragment(s) were characterized via PAGE and MALDI-TOF to confirm that the oligonucleotide fragment(s) contained a 5'-phosphorylated 8-oxoG. Furthermore, the yields for this stalling varied from app. 5-30% with 8-oxoG located at different positions and in different sequences. To gain a better understanding of the decreased nuclease efficiency, we probed: 1) H-bonding and spatial constraints; 2) anti-syn conformational changes; 3) concentration of divalent cation; and 4) secondary structure. This was carried out by introducing methylated or brominated purines (m1G, m6,6A, or 8-BrG), probing varying [Mg2+], and using circular dichroism (CD) to explore the formation of structured RNA. It was determined that spatial constraints imposed by conformational changes around the glycosidic bond may be partially responsible for stalling, however, the results do not fully explain some of the observed higher stalling yields. We hypothesize that altered π-π stacking along with induced H-bonding interactions between 8-oxoG and residues within the binding site may also play a role in the decreased Xrn-1 efficiency. Overall, these observations suggest that other factors, yet to be discovered/established, are likely to contribute to the decay of oxidized RNA. In addition, Xrn-1 degraded RNA containing m1G, and stalled mildly at sites where it encountered m6,6A, or 8-BrG, which is of particular interest given that the former two are naturally occurring modifications.

Stress Granule-Mediated Oxidized RNA Decay in P-Body: Hypothetical Role of ADAR1, Tudor-SN, and STAU1.

Reactive oxygen species (ROS) generated under oxidative stress (OS) cause oxidative damage to RNA. Recent studies have suggested a role for oxidized RNA in several human disorders. Under the conditions of oxidative stress, mRNAs released from polysome dissociation accumulate and initiate stress granule (SG) assembly. SGs are highly enriched in mRNAs, containing inverted repeat (IR) Alus in 3' UTRs, AU-rich elements, and RNA-binding proteins. SGs and processing bodies (P-bodies) transiently interact through a docking mechanism to allow the exchange of RNA species. However, the types of RNA species exchanged, and the mechanisms and outcomes of exchange are still unknown. Specialized RNA-binding proteins, including adenosine deaminase acting on RNA (ADAR1-p150), with an affinity toward inverted repeat Alus, and Tudor staphylococcal nuclease (Tudor-SN) are specifically recruited to SGs under OS along with an RNA transport protein, Staufen1 (STAU1), but their precise biochemical roles in SGs and SG/P-body docking are uncertain. Here, we critically review relevant literature and propose a hypothetical mechanism for the processing and decay of oxidized-RNA in SGs/P-bodies, as well as the role of ADAR1-p150, Tudor-SN, and STAU1.

Products of lipid, protein and RNA oxidation as signals and regulators of gene expression in plants.

Reactive oxygen species (ROS) are engaged in several processes essential for normal cell functioning, such as differentiation, anti-microbial defense, stimulus sensing and signaling. Interestingly, recent studies imply that cellular signal transduction and gene regulation are mediated not only directly by ROS but also by the molecules derived from ROS-mediated oxidation. Lipid peroxidation leads to non-enzymatic formation of oxylipins. These molecules were shown to modulate expression of signaling associated genes including genes encoding phosphatases, kinases and transcription factors. Oxidized peptides derived from protein oxidation might be engaged in organelle-specific ROS signaling. In turn, oxidation of particular mRNAs leads to decrease in the level of encoded proteins and thus, contributes to the post-transcriptional regulation of gene expression. Present mini review summarizes latest findings concerning involvement of products of lipid, protein and RNA oxidation in signal transduction and gene regulation.

Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells.

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress.