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Cells remodel splicing and translation machineries to mount specialized gene expression responses to stress. Here, we show that hypoxic human cells in 2D and 3D culture models increase the relative abundance of a longer mRNA variant of ribosomal protein S24 (RPS24L) compared to a shorter mRNA variant (RPS24S) by favoring the inclusion of a 22 bp cassette exon. Mechanistically, RPS24L and RPS24S are induced and repressed, respectively, by distinct pathways in hypoxia: RPS24L is induced in an autophagy-dependent manner, while RPS24S is reduced by mTORC1 repression in a hypoxia-inducible factor-dependent manner. RPS24L produces a more stable protein isoform that aids in hypoxic cell survival and growth, which could be exploited by cancer cells in the tumor microenvironment., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Neuropeptide Y (NPY) is an abundant neurohormone in human breast carcinomas that acts on a class of G-protein coupled receptors, of which NPY1R and NPY5R are the most highly expressed. This abundance is exploited for cancer imaging, but there is interest in pharmacological inhibition of the NPYRs to interrogate their functional relevance in breast cancer. We previously reported that NPY1R and NPY5R mRNA abundance is increased by hypoxia inducible factors, which sensitizes these receptors to NPY stimulation leading to enhanced migration and proliferation., Methods/results: Here, we measured the effects of NPY1R and NPY5R antagonists in normoxia and hypoxia on migration, proliferation, invasion, and signaling in 2D and 3D models of breast cancer cell lines MDA-MB-231 and MCF7. Antagonizing NPY1R and/or NPY5R in hypoxia compared to normoxia more greatly reduced MAPK signaling, cell proliferation, cell migration and invasion, and spheroid growth and invasion. The estrogen receptor positive MCF7 cells were significantly less invasive in 3D spheres when NPY5R was specifically inhibited. There were some discrepancies in the responses of each cell line to the isoform-specific antagonists and oxygen availability, therefore further investigations are required to dissect the intricacies of NPYR signaling dynamics. In human breast tumor tissue, we show via immunofluorescence that NPY5R protein levels and colocalization with hypoxia correlate with advanced cancer, and NPY1R protein correlates with adverse outcomes., Conclusions: Antagonizing the NPYRs has been implicated as a treatment for a wide variety of diseases. Therefore, these antagonists may aid in the development of novel cancer therapeutics and patient-based treatment plans., (© 2023. The Author(s).)

Spheroids enable the study of tumors and tumor hypoxia using a more representative model of the physiological environment compared to 2D cell culture. Spheroids can be grown in a cell suspension or when adhered to a solid scaffold. The spheroid formation method used is dependent on cell type. Here we describe the most common spheroid formation methods, including hanging drop, low adhesion plates, hydrogel, micropatterned plates, and microfluidics. After spheroids are formed, they can be used for drug treatment trials and analyzed using Western Blots, qPCR, and microscopy. Microscopy can then be used to measure the invasiveness of cells when a basement membrane is added to spheroids and for monitoring changes in the proliferation, quiescent, and necrotic zones of spheroids., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The hypoxia-inducible factors (HIF)-1α and HIF-2α are central regulators of transcriptional programmes in settings such as development and tumour expansion. HIF-2α moonlights as a cap-dependent translation factor. We provide new insights into how the interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, and the HIFs regulate one another in hypoxia and interferon-induced cells. We show that upon ISGylation induction and HIF-α stabilization, both HIFs promote protein ISGylates through transcriptional and/or post-transcriptional pathways. We show the first evidence of HIF-2α modification by ISG15. ISGylation induces system-level alterations to the HIF transcriptional programme and increases the cytoplasmic/nuclear fraction and translation activity of HIF-2α. This work identifies ISG15 as a regulator of hypoxic mRNA translation, which has implications for immune processes and disease progression., (© 2022 Federation of European Biochemical Societies.)

Neuropeptide Y (NPY) is an abundant neurohormone in the central and peripheral nervous system involved in feeding behavior, energy balance, nociception, and anxiety. Several NPY receptor (NPYR) subtypes display elevated expression in many cancers including in breast tumors where it is exploited for imaging and diagnosis. Here, we address how hypoxia, a common feature of the tumor microenvironment, influences the expression of the NPYRs. We show that NPY1R and NPY5R mRNA abundance is induced by hypoxia in a hypoxia inducible factor (HIF)-dependent manner in breast cancer cell lines MCF7 and MDA-MB-231. We demonstrate that HIFs bind to several genomic regions upstream of the NPY1R and NPY5R transcription start sites. In addition, the MAPK/ERK pathway is activated more rapidly upon NPY5R stimulation in hypoxic cells compared with normoxic cells. This pathway requires insulin-like growth factor 1 receptor (IGF1R) activity in normoxia, but not in hypoxic cells, which display resistance to the radiosensitizer and IGF1R inhibitor AG1024. Furthermore, hypoxic cells proliferate and migrate more when stimulated with NPY relative to normoxic cells and exhibit a more robust response to a Y5-specific agonist. Our data suggest that hypoxia-induced NPYRs render hypoxic cells more sensitive to NPY stimulation. Considering that breast tissue receives a constant supply of NPY, hypoxic breast tumors are the perfect storm for hyperactive NPYR. This study not only highlights a new relationship between the HIFs and NPYR expression and activity but may inform the use of chemotherapeutics targeting NPYRs and hypoxic cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Global translational remodeling has emerged as a principal mechanism of biological adaptation. Oxygen deficiency (hypoxia) disables the basal protein synthesis machinery ('Jekyll') and activates a hypoxic translational architecture ('Hyde') to drive translatome remodeling. Independent from mRNA-level fluctuations, this newer paradigm modernizes a field traditionally dominated by the hypoxia-inducible factor (HIF) transcriptional program., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

Ribosomes were once considered static in their composition because of their essential role in protein synthesis and kingdom-wide conservation. The existence of tolerated mutations in select ribosomal proteins (RPs), such as in Diamond-Blackfan anemia, is evidence that not all ribosome components are essential. Heterogeneity in the protein composition of eukaryotic ribosomes is an emerging concept with evidence that different pools of ribosomes exist with transcript-specificity. Here, we show that the polysome association of ribosomal proteins is altered by low oxygen (hypoxia), a feature of the tumor microenvironment, in human cells. We quantified ribosomal protein abundance in actively translating polysomes of normoxic and hypoxic HEK293 cells by tandem mass tags mass spectrometry. Our data suggest that RPS12 (eS12) is enriched in hypoxic monosomes, which increases the heavy polysome association of structured transcripts APAF-1 and XIAP. Furthermore, hypoxia induced five alternative splicing events within a subset of RP mRNAs in cell lines. One of these events in RPS24 (eS24 protein) alters the coding sequence to produce two protein isoforms that can incorporate into ribosomes. This splicing event is greatly induced in spheroids and correlates with tumor hypoxia in human prostate cancer. Our data suggest that hypoxia may influence the composition of the human ribosome through changes in RP incorporation and the production of hypoxia-specific RP isoforms., (© 2020 Brumwell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Hypoxia is a deficiency in oxygen delivery to tissues and is connected to physiological and pathophysiological processes such as embryonic development and cancer. The master regulators of oxygen homeostasis in mammalian cells are the heterodimeric hypoxia-inducible transcription factors 1 and 2 (HIF-1 and HIF-2, respectively). The oxygen-labile HIF-2α subunit has been implicated not only in transcription but also as a regulator of eukaryotic initiation factor 4E2 (eIF4E2)-directed hypoxic translation. Here, we have identified the DEAD box protein family member DDX28 as an interactor and negative regulator of HIF-2α that suppresses HIF-2α's ability to activate eIF4E2-directed translation. Stable silencing of DDX28 via short hairpin RNA (shRNA) in hypoxic human U87MG glioblastoma cells caused an increase of eIF4E2 binding to the m 7 GTP cap structure and the translation of eIF4E2 target mRNAs (including the HIF-2α mRNA itself). DDX28 depletion elevated nuclear and cytoplasmic HIF-2α protein, but HIF-2α transcriptional activity did not increase, possibly due to its already high nuclear abundance in hypoxic control cells. Depletion of DDX28 conferred a proliferative advantage to hypoxic, but not normoxic, cells. DDX28 protein levels are reduced in several cancers, including gliomas, relative to levels in normal tissue. Therefore, we uncover a regulatory mechanism for this potential tumor suppressor in the repression of HIF-2α- and eIF4E2-mediated translation activation of oncogenic mRNAs., (Copyright © 2020 American Society for Microbiology.)

Multicellular organisms balance oxygen delivery and toxicity by having oxygen pass through several barriers before cellular delivery. In human cell culture, these physiologic barriers are removed, exposing cells to higher oxygen levels. Human cells cultured in ambient air may appear normal, but this is difficult to assess without a comparison at physiologic oxygen. Here, we examined the effects of culturing human cells throughout the spectrum of oxygen availability on oxidative damage to macromolecules, viability, proliferation, the antioxidant and DNA damage responses, metabolism, and mitochondrial fusion and morphology. We surveyed 4 human cell lines cultured for 3 d at 7 oxygen conditions between 1 and 21% O 2 . We show that oxygen levels and cellular benefit are not inversely proportional, but the benefit peaks within the physioxic range. Normoxic cells are in a perpetual state of responding to damaged macromolecules and mitochondrial networks relative to physioxic cells, which could compromise an investigation. These data contribute to the concept of an optimal oxygen availability for cell culture in the physioxic range where the oxygen is not too high to reduce oxidative damage, and not too low for efficient oxidative metabolism, but just right: the Goldiloxygen zone.-Timpano, S., Guild, B. D., Specker, E. J., Melanson, G., Medeiros, P. J., Sproul, S. L. J., Uniacke, J. Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hypoxia is an aspect of the tumor microenvironment that is linked to radiation and chemotherapy resistance, metastasis, and poor prognosis. The ability of hypoxic tumor cells to achieve these cancer hallmarks is, in part, due to changes in their gene expression profiles. Cancer cells have a high demand for protein synthesis, and translational control is subsequently deregulated. Various mechanisms of translation initiation are active to improve the translation efficiency of select transcripts to drive cancer progression. This review will focus on a noncanonical cap-dependent translation initiation mechanism that utilizes the eIF4E homolog eIF4E2, a hypoxia-activated cap-binding protein that is implicated in hypoxic cancer cell migration, invasion, and tumor growth in mouse xenografts. A historical perspective about eIF4E2 and its various aliases will be provided followed by an evaluation of potential therapeutic strategies. The recent successes of disabling canonical translation and eIF4E with drugs should highlight the novel therapeutic potential of targeting the homologous eIF4E2 in the treatment of hypoxic solid tumors.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m 7 GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4F H ), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m 7 GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4F(H). O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2015. Published by The Company of Biologists Ltd.)

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.

Human tumors display considerable diversity in their genetic makeup but share common physiologic attributes such as a hypoxic microenvironment that contribute to the malignant phenotype. Hypoxic cells switch from eukaryotic initiation factor 4E (eIF4E) to eIF4E2 cap-dependent translation to synthesize a portion of their proteins. Here, we show that genetically distinct human cancer cells exploit eIF4E2-directed protein synthesis to form cellular masses larger than approximately 0.15 mm, the diffusion limit of oxygen. Cancer cells depleted of eIF4E2 are indistinguishable from control cells under normoxic conditions, but are unable to survive and proliferate in low oxygen conditions. Activation of eIF4E2-directed translation is essential for cancer cells to form a hypoxic tumor core in in vitro spheroids and to form detectable tumors in in vivo xenograft assays. In contrast, the eIF4E-directed protein synthesis pathway alone cannot sustain cellular adaptation to hypoxia in vitro or confer tumorigenic potential in xenograft assays. These data demonstrate that the phenotypic expression of the cancer genome requires translation by the eIF4E2-directed hypoxic protein synthesis machinery., (©2014 AACR)

The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate-positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus.

Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7-methylguanosine (m(7)-GpppG) 5' cap of messenger RNAs. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms. Although the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. We show that hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA-binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array of mRNAs, including that encoding the epidermal growth factor receptor. Once assembled at the rHRE, the HIF-2α-RBM4-eIF4E2 complex captures the 5' cap and targets mRNAs to polysomes for active translation, thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program by which oxygen tension switches the basic translation initiation machinery.

Here we describe how to use fluorescence in situ hybridization and immunofluorescence staining to determine the in situ distributions of specific mRNAs and proteins in Chlamydomonas reinhardtii. This unicellular eukaryotic green alga is a major model organism in cell biological research. Chlamydomonas is well suited for these approaches because one can determine the cytological location of fluorescence signals within a characteristic cellular anatomy relative to prominent cytological markers. Moreover, FISH and IF staining offer practical alternatives to techniques involving fluorescent proteins, which are difficult to express and detect in Chlamydomonas. The main goal of this review is to describe these powerful tools and to facilitate their routine use in Chlamydomonas research.

The compartmentalization of eukaryotic cells requires that newly synthesized proteins be targeted to the compartments in which they function. In chloroplasts, a few thousand proteins function in photosynthesis, expression of the chloroplast genome, and other processes. Most chloroplast proteins are synthesized in the cytoplasm, imported, and then targeted to a specific chloroplast compartment. The remainder are encoded by the chloroplast genome, synthesized within the organelle, and targeted by mechanisms that are only beginning to be elucidated. We used fluorescence confocal microscopy to explore the targeting mechanisms used by several chloroplast proteins in the green alga Chlamydomonas. These include the small subunit of ribulose bisphosphate carboxylase (rubisco) and the light-harvesting complex II (LHCII) subunits, which are imported from the cytoplasm, and 2 proteins synthesized in the chloroplast: the D1 subunit of photosystem II and the rubisco large subunit. We determined whether the targeting of each protein involves localized translation of the mRNA that encodes it. When this was the case, we explored whether the targeting sequence was in the nascent polypeptide or in the mRNA, based on whether the localization was translation-dependent or -independent, respectively. The results reveal 2 novel examples of targeting by localized translation, in LHCII subunit import and the targeting of the rubisco large subunit to the pyrenoid. They also demonstrate examples of each of the three known mechanisms-posttranslational, cotranslational (signal recognition particle-mediated), and mRNA-based-in the targeting of specific chloroplast proteins. Our findings can help guide the exploration of these pathways at the biochemical level.

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.

Many proteins of the photosynthesis complexes are encoded by the genome of the chloroplast and synthesized by bacterium-like ribosomes within this organelle. To determine where proteins are synthesized for the de novo assembly and repair of photosystem II (PSII) in the chloroplast of Chlamydomonas reinhardtii, we used fluorescence in situ hybridization, immunofluorescence staining, and confocal microscopy. These locations were defined as having colocalized chloroplast mRNAs encoding PSII subunits and proteins of the chloroplast translation machinery specifically under conditions of PSII subunit synthesis. The results revealed that the synthesis of the D1 subunit for the repair of photodamaged PSII complexes occurs in regions of the chloroplast with thylakoids, consistent with the current model. However, for de novo PSII assembly, PSII subunit synthesis was detected in discrete regions near the pyrenoid, termed T zones (for translation zones). In two PSII assembly mutants, unassembled D1 subunits and incompletely assembled PSII complexes localized around the pyrenoid, where we propose that they mark an intermediate compartment of PSII assembly. These results reveal a novel chloroplast compartment that houses de novo PSII biogenesis and the regulated transport of newly assembled PSII complexes to thylakoid membranes throughout the chloroplast.