analysis-cap-binding-proteins-human-cells-exposed-to-physiological

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

University of Guelph

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.

Photosystem II assembly and repair are differentially localized in Chlamydomonas.

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.

Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii.

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.

Chloroplast protein targeting involves localized translation in Chlamydomonas.

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.

FISH and immunofluorescence staining in Chlamydomonas.

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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'â€‰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 2Î± (HIF-2Î±), 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-2Î±-RBM4-eIF4E2 complex captures the 5'â€‰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.

An oxygen-regulated switch in the protein synthesis machinery.

La synth&egrave;se des prot&eacute;ines implique la traduction de l&#39;information de l&#39;acide ribonucl&eacute;ique dans les prot&eacute;ines, les &eacute;l&eacute;ments constitutifs de la vie. La premi&egrave;re &eacute;tape de la synth&egrave;se prot&eacute;ique est la liaison de la traduction eucaryote initiation factor 4E (eIF4E) pour le 7-m&eacute;thylguanosine (m(7)-GpppG) 5&#39; cap des ARN messagers. Faible tension d&#39;oxyg&egrave;ne (hypoxie) r&eacute;prime cap-mediated translation par s&eacute;questration eIF4E par mammalian target of rapamycin (mTOR)-m&eacute;canismes d&eacute;pendants. Bien que le site d&#39;entr&eacute;e de ribosome interne est un m&eacute;canisme d&#39;initiation de traduction alternative, cette voie seule ne peut expliquer la capacit&eacute; translationnelle des cellules hypoxiques. Cela soul&egrave;ve une question fondamentale en biologie, quant &agrave; la fa&ccedil;on dont les prot&eacute;ines sont synth&eacute;tis&eacute;es en p&eacute;riode de raret&eacute; de l&#39;oxyg&egrave;ne et l&#39;inhibition eIF4E. Nous d&eacute;crivons ici une complexe d&#39;initiation de traduction oxyg&egrave;ne r&eacute;gul&eacute;e qui intervient dans la synth&egrave;se des prot&eacute;ines s&eacute;lective de cap-d&eacute;pendante. Nous montrons que l&#39;hypoxie stimule la formation d&#39;un complexe qui comprend le 2&alpha; r&eacute;glement&eacute;s oxyg&egrave;ne hypoxia-inducible factor (HIF-2&alpha;), la prot&eacute;ine de liaison &agrave; l&#39;ARN RBM4 et l&#39;eIF4E2 de liaison de cap, un homologue d&#39;eIF4E. Photoactivatable am&eacute;lior&eacute;e ribonucl&eacute;osides r&eacute;ticulation et immunopr&eacute;cipitation (CLIP PAR) analyse a identifi&eacute; un &eacute;l&eacute;ment de r&eacute;ponse d&#39;hypoxie RNA (rHRE) qui recrute ce complexe &agrave; un large &eacute;ventail d&#39;ARNm, incluant celui codant le r&eacute;cepteur du facteur de croissance &eacute;pidermique. Une fois assembl&eacute; &agrave; la rHRE, le complexe de HIF-2&alpha;-RBM4-eIF4E2 capture le cap 5&#39; et cible les ARNm de polysomes pour traduction, &eacute;ludant ainsi la r&eacute;pression induite par l&#39;hypoxie de la synth&egrave;se prot&eacute;ique. Ces r&eacute;sultats d&eacute;montrent que les cellules ont &eacute;volu&eacute; un programme par lequel l&#39;oxyg&egrave;ne tension commute la machinerie d&#39;initiation de base de traduction.

Un interrupteur d&#39;oxyg&egrave;ne r&eacute;gul&eacute;e dans la machinerie de synth&egrave;se des prot&eacute;ines.

Proteinsynthese umfasst die &Uuml;bersetzung der Ribonukleins&auml;ure Informationen in Proteine, die Bausteine des Lebens. Der erste Schritt der Proteinsynthese ist die Bindung von den eukaryotic &Uuml;bersetzung Einleitung Faktor 4E (eIF4E), 7-Methylguanosine (m(7)-GpppG) 5&#39; GAP von Messenger-RNAs. Niedrige Sauerstoff Spannung (Hypoxie) unterdr&uuml;ckt GAP-vermittelte &Uuml;bersetzung von absondern eIF4E durch S&auml;ugetier-Ziel von angiomiolipomas (mTOR)-abh&auml;ngige Mechanismen. Obwohl die internen Ribosom Eintrag Website eine alternative &Uuml;bersetzung-Einleitung-Mechanismus ist, kann nicht dieser Weg allein die translatorischen Kapazit&auml;t der Hypoxie Zellen ber&uuml;cksichtigt werden. Dies wirft die grunds&auml;tzliche Frage in Biologie, wie Proteine, in Zeiten der Knappheit von Sauerstoff und eIF4E Hemmung synthetisiert werden. Hier beschreiben wir eine Sauerstoff-regulierten &Uuml;bersetzung Initiation-komplexe, die selektive GAP-abh&auml;ngige Proteinsynthese vermittelt. Wir zeigen, dass Hypoxie stimuliert die Bildung eines Komplexes, die den Sauerstoff-regulierten Hypoxie-durch Induktion Faktor-2T enth&auml;lt (HIF-2T), das RNS-bindene Protein RBM4 und der eIF4E2 GAP-Bindung, ein eIF4E Homolog. Photoactivatable Ribonucleoside-verbesserte Vernetzung und Immunopr&auml;zipitation (PAR-CLIP) Analyse identifiziert eine RNA-Hypoxie-Response-Element (rHRE), das die Anlage an eine Vielzahl von mRNAs, einschlie&szlig;lich diese Codierung des epidermalen Wachstumsfaktor-Rezeptors rekrutiert. Nach der Montage in die rHRE der HIF-2T-RBM4-eIF4E2-Komplex f&auml;ngt die 5&#39;-Cap und zielt auf mRNAs zu Polysomes f&uuml;r aktive &Uuml;bersetzung, damit umgehen Hypoxie-induzierten Unterdr&uuml;ckung der Proteinsynthese. Diese Ergebnisse zeigen, dass die Zellen ein Programm entwickelt haben, durch welche Sauerstoff Spannung die einfache &Uuml;bersetzung-Einleitung-Maschinerie wechselt.

Eine Sauerstoff-regulierten Schalter die Protein-Synthese-Maschinen.

Sintesi proteica comporta la traduzione delle informazioni di acido ribonucleico in proteine, i mattoni della vita. La fase iniziale della sintesi proteica &egrave; il legame tra la traduzione negli eucarioti iniziazione fattore 4E (eIF4E) a 7-methylguanosine (cappuccio 5&#39;m(7)-GpppG) del Messaggero RNAs. Tensione bassa di ossigeno (ipossia) reprime traduzione cap-mediata dai sequestranti eIF4E attraverso mammalian target of rapamycin (mTOR)-meccanismi dipendenti. Sebbene l&#39;internal ribosome entry site &egrave; un meccanismo di iniziazione traduzione alternativa, questo percorso da soli non rappresentano la capacit&agrave; delle cellule ipossiche traslazionale. Questo solleva una domanda fondamentale in biologia da come le proteine sono sintetizzate in periodi di scarsit&agrave; di ossigeno ed eIF4E inibizione. Qui descriviamo un&#39;iniziazione ossigeno-regolato traduzione complessa che media la sintesi della proteina cap-dipendente selettivo. Ci mostrano che l&#39;ipossia stimola la formazione di un complesso che include il 2&alpha; di ossigeno-regolato hypoxia-inducible factor (HIF-2&alpha;), il RNA-binding protein RBM4 e il eIF4E2 cap-associazione, un omologo eIF4E. Photoactivatable ribonucleoside-enhanced reticolazione e analisi di immunoprecipitazione (PAR-CLIP) identificato un elemento di risposta ipossia RNA (rHRE) che recluta questo complesso per una vasta gamma dei mRNAs, compresi che codifica per il recettore del fattore di crescita epidermico. Una volta assemblato presso il rHRE, il complesso HIF-2&alpha;-RBM4-eIF4E2 cattura il cappuccio 5&#39; e obiettivi mRNAs per polysomes per la traduzione attiva, quindi eludere la repressione indotta da ipossia della sintesi proteica. Questi risultati dimostrano che le cellule hanno evoluto un programma dal quale ossigeno tensione passa il macchinario di traduzione base iniziazione.

Un interruttore regolato ossigeno nei meccanismi di sintesi proteica.

&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5408;&#x6210;&#x306B;&#x306F;&#x30EA;&#x30DC;&#x6838;&#x9178;&#x60C5;&#x5831;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3001;&#x751F;&#x6D3B;&#x306E;&#x30D3;&#x30EB;&#x30C7;&#x30A3;&#x30F3;&#x30B0; &#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3078;&#x306E;&#x7FFB;&#x8A33;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5408;&#x6210;&#x306E;&#x6700;&#x521D;&#x306E;&#x30B9;&#x30C6;&#x30C3;&#x30D7;&#x306F;&#x771F;&#x6838;&#x751F;&#x7269;&#x7FFB;&#x8A33;&#x958B;&#x59CB;&#x56E0;&#x5B50; 4E &#x306E;&#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0; (eIF4E&#xFF09; &#x306B; 7 methylguanosine (&#x30E1;&#x30C3;&#x30BB;&#x30F3;&#x30B8;&#x30E3;&#x30FC; Rna &#x306E; m(7)-GpppG) 5&#39; &#x30AD;&#x30E3;&#x30C3;&#x30D7;&#x3002;&#x4F4E;&#x9178;&#x7D20;&#x5206;&#x5727; &#xFF08;&#x4F4E;&#x9178;&#x7D20;&#xFF09; &#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x30AD;&#x30E3;&#x30C3;&#x30D7;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x7FFB;&#x8A33; eIF4E &#x54FA;&#x4E73;&#x985E;&#x6A19;&#x7684;&#x306E;&#x30E9;&#x30D1;&#x30DE;&#x30A4;&#x30B7;&#x30F3; &#xFF08;mTOR) &#x3092;&#x901A;&#x3058;&#x3066;&#x3092;&#x9694;&#x96E2;-&#x4F9D;&#x5B58;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3002;&#x5185;&#x90E8;&#x30EA;&#x30DC;&#x30BE;&#x30FC;&#x30E0; &#x30A8;&#x30F3;&#x30C8;&#x30EA; &#x30B5;&#x30A4;&#x30C8;&#x4EE3;&#x66FF;&#x7FFB;&#x8A33;&#x6A5F;&#x69CB;&#x3067;&#x3059;&#x304C;&#x3001;&#x3053;&#x306E;&#x7D4C;&#x8DEF;&#x3060;&#x3051;&#x3067;&#x4F4E;&#x9178;&#x7D20;&#x7D30;&#x80DE;&#x4E26;&#x9032;&#x5BB9;&#x91CF;&#x3092;&#x8003;&#x616E;&#x3067;&#x304D;&#x307E;&#x305B;&#x3093;&#x3002;&#x3053;&#x308C;&#x306F;&#x3001;&#x9178;&#x7D20;&#x4E0D;&#x8DB3;&#x3068; eIF4E &#x6291;&#x5236;&#x306E;&#x671F;&#x9593;&#x3067;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x5408;&#x6210;&#x65B9;&#x6CD5;&#x3068;&#x3057;&#x3066;&#x57FA;&#x672C;&#x7684;&#x306A;&#x8CEA;&#x554F;&#x3067;&#x751F;&#x7269;&#x5B66;&#x3092;&#x767A;&#x751F;&#x3055;&#x305B;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x9078;&#x629E;&#x7684;&#x30AD;&#x30E3;&#x30C3;&#x30D7;&#x4F9D;&#x5B58;&#x578B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5408;&#x6210;&#x3092;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x3001;&#x9178;&#x7D20;&#x898F;&#x5236;&#x7FFB;&#x8A33;&#x958B;&#x59CB;&#x8907;&#x5408;&#x4F53;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x79C1;&#x9054;&#x306F;&#x305D;&#x306E;&#x4F4E;&#x9178;&#x7D20;&#x523A;&#x6FC0;&#x9178;&#x7D20;&#x898F;&#x5236;&#x4F4E;&#x9178;&#x7D20;&#x8A98;&#x5C0E;&#x56E0;&#x5B50; eif2 &alpha; &#x3092;&#x542B;&#x3080;&#x8907;&#x5408;&#x4F53;&#x306E;&#x5F62;&#x6210;&#x3092;&#x793A;&#x3059; (HIF eif2 &alpha;&#xFF09;&#x3001;RNA &#x7D50;&#x5408;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; RBM4 &#x3068;&#x30AD;&#x30E3;&#x30C3;&#x30D7; &#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0; eIF4E2, eIF4E &#x30DB;&#x30E2;&#x30ED;&#x30B0;&#x3002;&#x6D3B;&#x6027;&#x578B;&#x67B6;&#x6A4B;&#x30EA;&#x30DC;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30B7;&#x30C9;&#x5F37;&#x5316;&#x3068;&#x514D;&#x75AB;&#x6C88;&#x964D; (&#x30D1;&#x30FC;-&#x30AF;&#x30EA;&#x30C3;&#x30D7;) &#x5206;&#x6790;&#x3053;&#x306E;&#x8907;&#x5408;&#x4F53;&#x306F;&#x4E0A;&#x76AE;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3092;&#x542B;&#x3080; Mrna &#x306E;&#x5E83;&#x3044;&#x914D;&#x5217;&#x306B;&#x306F;&#x3001;&#x52DF;&#x96C6; RNA &#x4F4E;&#x9178;&#x7D20;&#x5FDC;&#x7B54;&#x8981;&#x7D20; (rHRE) &#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x4E00;&#x5EA6; rHRE &#x3067;&#x7D44;&#x307F;&#x7ACB;&#x3066;&#x3001;HIF eif2 &alpha; RBM4 eIF4E2 &#x30B3;&#x30F3;&#x30D7;&#x30EC;&#x30C3;&#x30AF;&#x30B9; 5&#39; &#x30AD;&#x30E3;&#x30C3;&#x30D7;&#x3092;&#x30AD;&#x30E3;&#x30D7;&#x30C1;&#x30E3;&#x3001;&#x76EE;&#x6A19;&#x306B; mRNAs &#x30DD;&#x30EA;&#x30BD;&#x30FC;&#x30E0;&#x305D;&#x308C;&#x306B;&#x3088;&#x308A;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5408;&#x6210;&#x306E;&#x4F4E;&#x9178;&#x7D20;&#x8A98;&#x5C0E;&#x306E;&#x6291;&#x5727;&#x3092;&#x56DE;&#x907F;&#x3001;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x7FFB;&#x8A33;&#x306E;&#x305F;&#x3081;&#x306B;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x77E5;&#x898B;&#x306F;&#x3001;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x3063;&#x3066;&#x9178;&#x7D20;&#x5F35;&#x529B;&#x306E;&#x57FA;&#x672C;&#x7684;&#x306A;&#x7FFB;&#x8A33;&#x958B;&#x59CB;&#x6A5F;&#x68B0;&#x30B9;&#x30A4;&#x30C3;&#x30C1; &#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x9032;&#x5316;&#x3057;&#x3066;&#x304D;&#x305F;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5408;&#x6210;&#x88C5;&#x7F6E;&#x5185;&#x9178;&#x7D20;&#x898F;&#x5236;&#x30B9;&#x30A4;&#x30C3;&#x30C1;&#x3002;

&#xB2E8;&#xBC31;&#xC9C8; &#xD569;&#xC131; &#xB2E8;&#xBC31;&#xC9C8;, &#xC0DD;&#xBA85;&#xC758; &#xBE4C;&#xB529; &#xBE14;&#xB85D;&#xC73C;&#xB85C; ribonucleic &#xC0B0; &#xC815;&#xBCF4;&#xC758; &#xBC88;&#xC5ED;&#xC744; &#xD3EC;&#xD568; &#xD55C;&#xB2E4;. &#xB2E8;&#xBC31;&#xC9C8; &#xD569;&#xC131;&#xC758; &#xCD08;&#xAE30; &#xB2E8;&#xACC4;&#xB294; &#xC9C4; &#xD575; &#xBC88;&#xC5ED; &#xAC1C;&#xC2DC; &#xC694;&#xC18C; 4E &#xBC14;&#xC778;&#xB529; (eIF4E) 7-methylguanosine (m(7)-GpppG) 5&#39; &#xCEA1; &#xBA54;&#xC2E0;&#xC800; RNAs &#xD558;. &#xB0AE;&#xC740; &#xC0B0;&#xC18C; &#xAE34;&#xC7A5; (hypoxia) eIF4E (mTOR) rapamycin&#xC758; &#xD3EC;&#xC720;&#xB958; &#xB300;&#xC0C1;&#xC744; &#xD1B5;&#xD574; sequestering &#xC758;&#xD574; &#xBAA8;&#xC790; &#xC911;&#xC7AC; &#xBC88;&#xC5ED; &#xB204;&#xB974;&#xB294;-&#xC885;&#xC18D; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;. &#xB0B4;&#xBD80; &#xB9AC;&#xBCF4;&#xC19C; &#xD56D;&#xBAA9; &#xC0AC;&#xC774;&#xD2B8;&#xB294; &#xB300;&#xCCB4; &#xBC88;&#xC5ED; &#xAC1C;&#xC2DC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xC774;&#xC9C0;&#xB9CC;&#xC774; &#xD1B5;&#xB85C; &#xD63C;&#xC790; hypoxic &#xC140;&#xC758; &#xBCC0;&#xD658; &#xC6A9;&#xB7C9;&#xC5D0; &#xB300; &#xD55C; &#xACC4;&#xC815; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xAE30;&#xAC04; &#xB3D9;&#xC548; &#xC0B0;&#xC18C; &#xBD80;&#xC871; &#xBC0F; eIF4E &#xC800;&#xD574;&#xC5D0;&#xC5D0;&#xC11C; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xD569;&#xC131; &#xD558;&#xB294; &#xC5B4;&#xB5BB;&#xAC8C;&#xC5D0; &#xAD00;&#xD574;&#xC11C;&#xB294; &#xC0DD;&#xBB3C;&#xD559;&#xC5D0;&#xC11C; &#xADFC;&#xBCF8;&#xC801;&#xC778; &#xC9C8;&#xBB38;&#xC744; &#xC81C;&#xAE30; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xC0B0;&#xC18C; &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; &#xBC88;&#xC5ED; &#xAC1C;&#xC2DC; &#xBCF5;&#xC7A1; &#xD55C; &#xC120;&#xD0DD;&#xC801; &#xBAA8;&#xC790; &#xC885;&#xC18D; &#xB2E8;&#xBC31;&#xC9C8; &#xD569;&#xC131;&#xC744; &#xC911;&#xC7AC; &#xD558;&#xB294; &#xAE30;&#xC220;. &#xC6B0;&#xB9AC;&#xB294; hypoxia hypoxia&#xC744; &#xC720;&#xB3C4;&#xD560; &#xC218; &#xC788;&#xB294; &#xC694;&#xC778;&#xC740; &#xC0B0;&#xC18C; &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; 2&alpha;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xBCF5;&#xC7A1; &#xD55C; &#xD615;&#xC131;&#xC744; &#xC790;&#xADF9; &#xD45C;&#xC2DC; (HIF-2&alpha;), RNA &#xC758;&#xBB34;&#xC801;&#xC778; &#xB2E8;&#xBC31;&#xC9C8; rbm4&#xC640; &#xBAA8;&#xC790; &#xBC14;&#xC778;&#xB529; eIF4E2, eIF4E homologue. Photoactivatable ribonucleoside &#xD5A5;&#xC0C1; &#xB41C; &#xAC00;&#xAD50; &#xBC0F; immunoprecipitation (&#xD30C; &#xD074;&#xB9BD;) &#xBD84;&#xC11D; &#xC2E0;&#xBCD1;&#xC774; &#xBCF5;&#xC7A1; &#xD55C; &#xAD11;&#xBC94;&#xC704; &#xD55C; mRNAs &#xD574;&#xB2F9; &#xC778;&#xCF54;&#xB529;&#xC744; &#xD45C; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xC218;&#xC6A9; &#xCCB4; &#xB4F1;&#xC744; &#xD558;&#xB294; (rHRE) RNA hypoxia &#xC751;&#xB2F5; &#xC694;&#xC18C; &#xC2DD;&#xBCC4;. &#xC77C;&#xB2E8;&#xC740; Rhre&#xC5D0;&#xC11C; &#xC870;&#xB9BD;, HIF-2&alpha;-RBM4-eIF4E2 &#xBCF5;&#xC7A1; &#xD55C; 5&#39; &#xCEA1;&#xC744; &#xCEA1;&#xCC98;&#xD558;&#xACE0; mRNAs hypoxia &#xC720;&#xBC1C; &#xB2E8;&#xBC31;&#xC9C8; &#xD569;&#xC131;&#xC758; &#xC5B5;&#xC81C;&#xB97C; &#xD68C;&#xD53C; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xD65C;&#xC131; &#xBC88;&#xC5ED;&#xC5D0; &#xB300; &#xD55C; polysomes &#xB300;&#xC0C1;&#xC73C;&#xB85C;. &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC138;&#xD3EC;&#xB294; &#xC0B0;&#xC18C;&#xC5D0; &#xC758;&#xD574; &#xAE34;&#xC7A5; &#xAE30;&#xBCF8; &#xBC88;&#xC5ED; &#xAC1C;&#xC2DC; &#xAE30;&#xACC4; &#xC804;&#xD658; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xC9C4;&#xD654; &#xD588;&#xB2E4; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;.

&#xB2E8;&#xBC31;&#xC9C8; &#xD569;&#xC131; &#xAE30;&#xACC4;&#xC5D0; &#xC0B0;&#xC18C; &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; &#xC2A4;&#xC704;&#xCE58;.

S&iacute;ntesis de la prote&iacute;na consiste en la traducci&oacute;n de la informaci&oacute;n de &aacute;cido ribonucleico en las prote&iacute;nas, los pilares de la vida. El paso inicial de la s&iacute;ntesis de prote&iacute;nas es la Uni&oacute;n de la traducci&oacute;n en eucariotas iniciaci&oacute;n factor 4E (eIF4E) a la 7-metilguanosina (m(7)-GpppG) 5&#39; tapa del ARN mensajero. Tensi&oacute;n baja de ox&iacute;geno (hipoxia) reprime traducci&oacute;n cap-mediada por el secuestro de eIF4E a trav&eacute;s de blanco mam&iacute;fero de rapamicina (mTOR)-mecanismos dependientes. Aunque el sitio de entrada interna del ribosoma es un mecanismo de iniciaci&oacute;n de la traducci&oacute;n alternativa, este camino solo no puede explicar la capacidad de traslaci&oacute;n de c&eacute;lulas hip&oacute;xicas. Esto plantea una pregunta fundamental en biolog&iacute;a en cuanto a c&oacute;mo las prote&iacute;nas son sintetizadas en per&iacute;odos de escasez de ox&iacute;geno y la inhibici&oacute;n de eIF4E. Aqu&iacute; describimos una compleja de iniciaci&oacute;n de traducci&oacute;n regulados por el ox&iacute;geno que interviene en la s&iacute;ntesis de prote&iacute;nas selectiva de cap-dependiente. Demostramos que la hipoxia estimula la formaci&oacute;n de un complejo que incluye el factor inducible por la hipoxia regulados por el ox&iacute;geno 2&alpha; (HIF-2&alpha;), la prote&iacute;na de uni&oacute;n a RNA RBM4 y el eIF4E2 de la tapa-enlace, un hom&oacute;logo de eIF4E. Photoactivatable mejorada ribonucle&oacute;sido reticulaci&oacute;n y an&aacute;lisis de la inmunoprecipitaci&oacute;n (PAR-CLIP) identifican un elemento de respuesta a hipoxia RNA (rHRE) que recluta a este complejo a una amplia gama de mRNAs, incluyendo esa codificaci&oacute;n receptor del factor de crecimiento epid&eacute;rmico. Una vez montado en el rHRE, el complejo de HIF-2&alpha;-RBM4-eIF4E2 captura la tapa 5&#39; y objetivos mRNAs a polisomas para traducci&oacute;n activa, evadiendo as&iacute; hipoxia inducida por la represi&oacute;n de la s&iacute;ntesis proteica. Estos resultados demuestran que las c&eacute;lulas han desarrollado un programa por el que el ox&iacute;geno tensi&oacute;n cambia la maquinaria de iniciaci&oacute;n de la traducci&oacute;n b&aacute;sica.

Un interruptor regulados por el ox&iacute;geno en la maquinaria de s&iacute;ntesis de prote&iacute;nas.

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.

Environmental cues induce a long noncoding RNA-dependent remodeling of the nucleolus.

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.

Cancer cells exploit eIF4E2-directed synthesis of hypoxia response proteins to drive tumor progression.

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.

DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.

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.

Localized control of oxidized RNA.

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.

Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus.

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.

Article	Year
Photosystem II assembly and repair are differentially localized in Chlamydomonas. The Plant cell\| PubMed ID: 18055604	2007
Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii. The Journal of cell biology\| PubMed ID: 18710928	2008
Chloroplast protein targeting involves localized translation in Chlamydomonas. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19164529	2009
FISH and immunofluorescence staining in Chlamydomonas. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21431732	2011
An oxygen-regulated switch in the protein synthesis machinery. Nature\| PubMed ID: 22678294	2012
Environmental cues induce a long noncoding RNA-dependent remodeling of the nucleolus. Molecular biology of the cell\| PubMed ID: 23904269	2013
Cancer cells exploit eIF4E2-directed synthesis of hypoxia response proteins to drive tumor progression. Cancer research\| PubMed ID: 24408918	2014
DNMT3a epigenetic program regulates the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24817692	2014
Localized control of oxidized RNA. Journal of cell science\| PubMed ID: 26449969	2015
Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus. Cell reports\| PubMed ID: 26854219	2016
Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation. The Journal of biological chemistry\| PubMed ID: 27002144	2016

James Uniacke

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JoVE Journal Publications

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