methods-to-investigate-regulatory-role-small-rnas-ribosomal-occupancy

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of <em>Plasmodium falciparum</em>

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Duke University School of Medicine

Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.

Out with the old, in with the new? Comparing methods for measuring protein degradation.

In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in P. falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.

Polysome profiling of the malaria parasite Plasmodium falciparum.

&#x643A;&#x5E26;&#x53D8;&#x5F02;&#x8840;&#x7EA2;&#x86CB;&#x767D;&#x57FA;&#x56E0; (HbS)&#xFF0C;&#x800C;&#x5BFC;&#x81F4;&#x9570;&#x72B6;&#x7EC6;&#x80DE;&#x75C5;&#x548C;&#x6297;&#x62D2;&#x7684;&#x6076;&#x6027;&#x759F;&#x75BE;&#x5BC4;&#x751F;&#x866B;&#x759F;&#x539F;&#x866B;&#x611F;&#x67D3;&#x7684;&#x7EA2;&#x7EC6;&#x80DE;&#x3002;&#x8FD9;&#x79CD;&#x963B;&#x529B;&#xFF0C;&#x957F;&#x671F;&#x4EE5;&#x6765;&#x88AB;&#x516C;&#x8BA4;&#x4E3A;&#x7EFC;&#x5408;&#x8BC4;&#x5224;&#xFF0C;&#x5176;&#x4E2D;&#x7684;&#x5206;&#x5B50;&#x57FA;&#x7840;&#x4ECD;&#x7136;&#x662F;&#x672A;&#x5B8C;&#x5168;&#x7406;&#x89E3;&#x7684;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x7684;&#x6C34;&#x5E73;&#x53CA; microRNA (&#x7C73;&#x5C14;) &#x7EC4;&#x6210;&#xFF0C;Hba &#x6742;&#x5408;&#x6027;&#x6216;&#x7EAF;&#x5408; HbSS &#x7EA2;&#x7EC6;&#x80DE;&#xFF0C;&#x6709;&#x52A9;&#x4E8E;&#x62B5;&#x6297;&#x759F;&#x539F;&#x866B;&#x3002;&#x671F;&#x95F4;&#x5E76;&#x759F;&#x539F;&#x866B;&#x7684;&#x751F;&#x547D;&#x5468;&#x671F;&#xFF0C;&#x7EA2;&#x7EC6;&#x80DE; Mirna &#x7684;&#x5B50;&#x96C6;&#x8F6C;&#x79FB;&#x5230;&#x8FD9;&#x79CD;&#x5BC4;&#x751F;&#x866B;&#x3002;&#x4E24;&#x4E2A; Mirna&#xFF0C;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 451 &#x548C;&#x8BA9;-7i &#x9AD8;&#x6D53;&#x7F29; Hba &#x548C; HbSS &#x7EA2;&#x7EC6;&#x80DE;&#x53CA;&#x8FD9;&#x4E9B; Mirna &#x4E0E;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9;-223&#xFF0C;&#x4E0D;&#x5229;&#x76D1;&#x7BA1;&#x759F;&#x539F;&#x866B;&#x751F;&#x957F;&#x3002;&#x5947;&#x602A;&#x7684;&#x662F;&#xFF0C;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#xFF0C;&#x548C;&#x5E73;&#x53F7;&#x7A7A;&#x95F4;&#x7AD9; 451 &#x548C;&#x8BA9; 7i &#x7EB3;&#x5165;&#x91CD;&#x8981;&#x5BC4;&#x751F;&#x866B;&#x4FE1;&#x4F7F; Rna&#xFF0C;&#x5E76;&#x901A;&#x8FC7;&#x53D7;&#x635F;&#x7684;&#x6838;&#x7CD6;&#x4F53;&#x52A0;&#x8F7D;&#xFF0C;&#x5BFC;&#x81F4;&#x8F6C;&#x5316;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x9570;&#x72B6;&#x7EC6;&#x80DE;&#x7EA2;&#x7EC6;&#x80DE;&#x8868;&#x73B0;&#x51FA;&#x7EC6;&#x80DE;&#x5185;&#x5728;&#x6297;&#x759F;&#x75BE;&#x90E8;&#x5206;&#x901A;&#x8FC7;&#x975E;&#x5178;&#x578B;&#x7C73;&#x5C14;&#x6D3B;&#x52A8;&#xFF0C;&#x8FD9;&#x53EF;&#x80FD;&#x662F;&#x590D;&#x6742;&#x7684;&#x771F;&#x6838;&#x75C5;&#x539F;&#x4F53;&#x552F;&#x4E00;&#x7684;&#x4E3B;&#x673A;&#x9632;&#x5FA1;&#x7B56;&#x7565;&#x3002;

&#x9570;&#x72B6;&#x7EC6;&#x80DE;&#x7EA2;&#x7EC6;&#x80DE; Microrna &#x5230;&#x6076;&#x6027;&#x759F;&#x539F;&#x866B;&#x6613;&#x4F4D;&#x6291;&#x5236;&#x5BC4;&#x751F;&#x866B;&#x7FFB;&#x8BD1;&#x5E76;&#x6709;&#x52A9;&#x4E8E;&#x62B5;&#x6297;&#x759F;&#x75BE;&#x3002;

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance.

&Eacute;rythrocytes portant un all&egrave;le variant de l&#39;h&eacute;moglobine (HbS), qui provoque la dr&eacute;panocytose et r&eacute;siste &agrave; l&#39;infection par le parasite Plasmodium falciparum de paludisme. La base mol&eacute;culaire de cette r&eacute;sistance, qui est reconnue depuis longtemps comme multifactorielle, reste incompl&egrave;tement comprise. Ici, nous montrons que la composition de microARN (miARN) dysr&eacute;gulation, HBA h&eacute;t&eacute;rozygotes ou homozygotes HbSS &eacute;rythrocytes, contribue &agrave; la r&eacute;sistance contre P. falciparum. Pendant la dur&eacute;e de vie d&#39;intra-&eacute;rythrocytaire de P. falciparum, un sous-ensemble des &eacute;rythrocytes miARN translocation dans le parasite. Deux miARN, miR-451 et laissez-7i, &eacute;tait hautement enrichi dans les &eacute;rythrocytes de HBA et HbSS, et ces miARN, avec miR-223, r&eacute;gul&eacute; n&eacute;gativement la croissance du parasite. &Eacute;tonnamment, nous avons constat&eacute; que miR-451 et let-7i int&eacute;gr&eacute;s dans les ARN messagers parasite essentiel et, par l&#39;interm&eacute;diaire de chargement ribosomique avec facult&eacute;s affaiblies, a provoqu&eacute; une inhibition translationnelle. Par cons&eacute;quent, dr&eacute;panocytose &eacute;rythrocytes pi&egrave;ce r&eacute;sistance cellulaire intrins&egrave;que au paludisme en partie gr&acirc;ce &agrave; une activit&eacute; atypique miRNA, qui peut repr&eacute;senter une strat&eacute;gie de d&eacute;fense du h&ocirc;te unique contre les complexes pathog&egrave;nes eucaryotes.

Translocation de dr&eacute;panocytose &eacute;rythrocytaire microARN dans Plasmodium falciparum inhibe la traduction de parasite et contribue &agrave; la r&eacute;sistance du paludisme.

Erythrozyten mit einem variant H&auml;moglobin Allel (HbS), Sichelzellan&auml;mie und str&auml;ubt sich gegen Infektion durch die Malaria Parasit Plasmodium Falciparum. Die molekulare Basis dieser Widerstand, der seit langem als multifaktorielle anerkannt hat, bleibt unvollst&auml;ndig verstanden. Hier zeigen wir, dass die defekten MicroRNA (MiRNA) Zusammensetzung, heterozygote HbAS oder Homozygote HbSS Erythrozyten, zum Widerstand gegen p. Falciparum beitr&auml;gt. W&auml;hrend des Lebenszyklus f&uuml;r die Intraerythrocytic von p. Falciparum eine Teilmenge der Erythrozyten MiRNAs akkumulieren in den Parasiten. Zwei MiRNAs, MiR-451 und lassen-7i, waren hoch angereichertes HbAS und HbSS Erythrozyten, und diese MiRNAs, zusammen mit MiR-223, negativ reguliert Parasit Wachstum. &Uuml;berraschend, wir gefunden, die MiR-451 und lassen-7i in wesentlichen Parasit Messenger-RNAs integriert und &uuml;ber gest&ouml;rte ribosomal laden, f&uuml;hrte translational Hemmung. Daher weisen Sichelzelle Erythrozyten Zelle-intrinsische Resistenz gegen Malaria Teil durch eine atypische MiRNA-Aktivit&auml;t, die eine einzigartige Host-Verteidigungsstrategie gegen komplexe eukaryotischen Krankheitserreger darstellen kann.

Translokation der Sichelzelle Erythrozyten MicroRNAs in Plasmodium Falciparum Parasit &Uuml;bersetzung hemmt und tr&auml;gt zur Malaria-Resistenz.

Eritrociti che trasportano un allele variante di emoglobina (HbS), che causa la malattia di cellule di falce e resiste all&#39;infezione con il parassita Plasmodium falciparum malaria. Le basi molecolari di questa resistenza, che &egrave; stata a lungo riconosciuta come multifattoriale, rimane non completamente capita. Qui vi mostriamo che la composizione microRNA (miRNA), HBA eterozigoti o omozigoti HbSS eritrociti, contribuisce alla resistenza contro p. falciparum. Durante il ciclo di vita di intraerythrocytic di p. falciparum, un sottoinsieme di eritrociti miRNAs traslocano in parassita. Due miRNAs, miR-451 e let-7i, erano altamente arricchito negli eritrociti HBA e HbSS, e questi miRNAs, insieme a miR-223, regolato negativamente la crescita del parassita. Sorprendentemente, abbiamo trovato che miR-451 e let-7i integrato nel parassita essenziale Messaggero RNAs e, tramite caricamento ribosomiale compromessa, ha provocato l&#39;inibizione traslazionale. Quindi, gli eritrociti cellule falciformi esibiscono cella-intrinseca resistenza alla malaria in parte attraverso un&#39;attivit&agrave; di miRNA atipico, che pu&ograve; rappresentare una strategia di difesa ospite unico contro agenti patogeni eucarioti complessi.

Traslocazione di falciforme degli eritrociti microRNA in Plasmodium falciparum inibisce la traduzione di parassita e contribuisce alla resistenza della malaria.

&#x8D64;&#x8840;&#x7403;&#x306F;&#x3001;&#x938C;&#x72B6;&#x8D64;&#x8840;&#x7403;&#x75C5;&#x306E;&#x539F;&#x56E0;&#x3057;&#x3001;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x539F;&#x866B;&#x306B;&#x3088;&#x308B;&#x611F;&#x67D3;&#x306B;&#x62B5;&#x6297;&#x3059;&#x308B;&#x30D0;&#x30EA;&#x30A2;&#x30F3;&#x30C8; &#x30D8;&#x30E2;&#x30B0;&#x30ED;&#x30D3;&#x30F3;&#x907A;&#x4F1D;&#x5B50; (HbS)&#x3002;&#x9577;&#x3044;&#x591A;&#x56E0;&#x5B50;&#x3068;&#x3057;&#x3066;&#x8A8D;&#x8B58;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;&#x3053;&#x306E;&#x62B5;&#x6297;&#x6027;&#x306E;&#x5206;&#x5B50;&#x57FA;&#x76E4;&#x306E;&#x4E0D;&#x5B8C;&#x5168;&#x306A;&#x7406;&#x89E3;&#x307E;&#x307E;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x6765;&#x3059;&#x30DE;&#x30A4;&#x30AF;&#x30ED; Rna &#xFF08;miRNA&#xFF09; &#x7D44;&#x6210;&#x306E;&#x30D8;&#x30C6;&#x30ED;&#x63A5;&#x5408;&#x4F53;&#x306E; Hba &#x307E;&#x305F;&#x306F;&#x30DB;&#x30E2; hbss &#x306E;&#x4EE3;&#x308A;&#x306E;&#x8D64;&#x8840;&#x7403;&#x306E;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x62B5;&#x6297;&#x6027;&#x306B;&#x5BC4;&#x4E0E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x306E;&#x8D64;&#x8840;&#x7403;&#x5BFF;&#x547D;&#x30B5;&#x30A4;&#x30AF;&#x30EB;&#x4E2D;&#x306B;&#x8D64;&#x8840;&#x7403; miRNAs &#x306E;&#x30B5;&#x30D6;&#x30BB;&#x30C3;&#x30C8;&#x306B;&#x5BC4;&#x751F;&#x866B;&#x3092;&#x5909;&#x3048;&#x307E;&#x3059;&#x3002;2 &#x3064;&#x306E; Mirna&#x3001;&#x30DF;&#x30FC;&#x30EB; 451 &#x3068;&#x805E;&#x304B;&#x305B;&#x3066; 7i &#x975E;&#x5E38;&#x306B; Hba &#x3068; hbss &#x306E;&#x4EE3;&#x308A;&#x306E;&#x8D64;&#x8840;&#x7403;&#x3067;&#x6FC3;&#x7E2E;&#x3055;&#x308C;&#x3001;&#x30DF;&#x30FC;&#x30EB;-223 &#x3068;&#x4E00;&#x7DD2;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E; miRNAs &#x306F;&#x5426;&#x5B9A;&#x7684;&#x306B;&#x5BC4;&#x751F;&#x866B;&#x6210;&#x9577;&#x3092;&#x898F;&#x5236;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x9A5A;&#x3044;&#x305F;&#x3053;&#x3068;&#x306B;&#x3001;&#x6211;&#x3005; &#x306F;&#x30DF;&#x30FC;&#x30EB; 451 &#x3092;&#x767A;&#x898B;&#x3057;&#x305F;&#x3068; let 7i &#x4E0D;&#x53EF;&#x6B20;&#x5BC4;&#x751F;&#x866B;&#x30E1;&#x30C3;&#x30BB;&#x30F3;&#x30B8;&#x30E3;&#x30FC; Rna &#x306B;&#x7D71;&#x5408;&#x3057;&#x3001;&#x969C;&#x5BB3;&#x306E;&#x30EA;&#x30DC;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x8AAD;&#x307F;&#x8FBC;&#x307F;&#x306B;&#x3088;&#x3063;&#x3066;&#x4E26;&#x9032;&#x963B;&#x5BB3;&#x306B;&#x8D77;&#x56E0;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x305D;&#x308C;&#x6545;&#x306B;&#x3001;&#x938C;&#x72B6;&#x8D64;&#x8840;&#x7403;&#x8D64;&#x8840;&#x7403;&#x7D30;&#x80DE;&#x56FA;&#x6709;&#x306E;&#x62B5;&#x6297;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x8907;&#x96D1;&#x306A;&#x771F;&#x6838;&#x751F;&#x7269;&#x306E;&#x75C5;&#x539F;&#x4F53;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x4E00;&#x610F;&#x306E;&#x30DB;&#x30B9;&#x30C8;&#x9632;&#x885B;&#x6226;&#x7565;&#x3092;&#x8868;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x975E;&#x5B9A;&#x578B; miRNA &#x6D3B;&#x52D5;&#x306E;&#x4E00;&#x90E8;&#x306F;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x306B;&#x5C55;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x938C;&#x72B6;&#x8D64;&#x8840;&#x7403;&#x8D64;&#x8840;&#x7403; microRNAs &#x306E;&#x8EE2;&#x6D41;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x539F;&#x866B;&#x306B;&#x306F;&#x5BC4;&#x751F;&#x866B;&#x306E;&#x7FFB;&#x8A33;&#x3092;&#x963B;&#x5BB3;&#x3057;&#x3066;&#x30DE;&#x30E9;&#x30EA;&#x30A2;&#x8010;&#x6027;&#x306B;&#x5BC4;&#x4E0E;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC801;&#xD608;&#xAD6C; &#xBCC0;&#xD615; &#xD5E4;&#xBAA8;&#xAE00;&#xB85C;&#xBE48; &#xB300;&#xB9BD; (HbS), &#xB0AB; &#xC138;&#xD3EC; &#xC9C8;&#xBCD1;&#xC758; &#xC6D0;&#xC778;&#xACFC; &#xB9D0;&#xB77C;&#xB9AC;&#xC544; &#xAE30;&#xC0DD;&#xCDA9; Plasmodium falciparum&#xC5D0; &#xC758;&#xD574; &#xAC10;&#xC5FC;&#xC744; &#xC800;&#xD56D; &#xD558;&#xB294; &#xC6B4;&#xBC18;. &#xC624;&#xB798; multifactorial&#xB85C; &#xC778;&#xC815; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;,&#xC774; &#xC800;&#xD56D;&#xC758; &#xBD84;&#xC790; &#xB2E8;&#xC704;&#xB85C; &#xB0A8;&#xC544; &#xBD88;&#xC644;&#xC804; &#xD558; &#xAC8C; &#xC774;&#xD574;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; dysregulated microRNA (&#xBBF8;&#xB974;) &#xC870;&#xC131; heterozygous Hba &#xB610;&#xB294; &#xC801;&#xD608;&#xAD6C;, HbSS homozygous P. falciparum&#xC5D0; &#xB300; &#xD55C; &#xC800;&#xD56D;&#xC5D0; &#xAE30;&#xC5EC;&#xB97C; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. P. falciparum intraerythrocytic &#xC218;&#xBA85; &#xC8FC;&#xAE30; &#xB3D9;&#xC548; &#xC801;&#xD608;&#xAD6C; Mirnas&#xC758; &#xD558;&#xC704; &#xC9D1;&#xD569; &#xAE30;&#xC0DD;&#xCDA9;&#xC73C;&#xB85C; &#xC774;&#xB3D9;. &#xB450; miRNAs, &#xBBF8;&#xB974;-451 &#xBC0F; &#xD558;&#xC790; 7i &#xD588;&#xB2E4; Hba &#xBC0F; HbSS &#xC801;&#xD608;&#xAD6C;&#xC5D0;&#xC11C; &#xB9E4;&#xC6B0; &#xD48D;&#xC131; &#xD558; &#xACE0; &#xBBF8;&#xB974;-223, &#xD568;&#xAED8; &#xC774;&#xB7EC;&#xD55C; Mirnas&#xB294; &#xBD80;&#xC815;&#xC801;&#xC778; &#xAE30;&#xC0DD;&#xCDA9; &#xC131;&#xC7A5; &#xADDC;&#xC81C;. &#xB180;&#xB78D;&#xAC8C;&#xB3C4;, &#xC6B0;&#xB9AC;&#xB294; &#xBBF8;&#xB974;-451&#xC744; &#xBC1C;&#xACAC; &#xD558; &#xACE0; &#xD558;&#xC790; 7i &#xD544;&#xC218; &#xAE30;&#xC0DD;&#xCDA9; &#xBA54;&#xC2E0;&#xC800; Rnas&#xC5D0; &#xD1B5;&#xD569; &#xD558; &#xACE0;, &#xC7A5;&#xC560;&#xC778;&#xB41C; ribosomal &#xB85C;&#xB4DC;&#xB97C; &#xD1B5;&#xD574; &#xBCC0;&#xD658; &#xC5B5;&#xC81C; &#xACB0;&#xACFC;. &#xB530;&#xB77C;&#xC11C;, &#xC801;&#xD608;&#xAD6C; &#xC801;&#xD608;&#xAD6C; &#xB9D0;&#xB77C;&#xB9AC;&#xC544; &#xBCF5;&#xC7A1; &#xD55C; &#xC9C4; &#xD575; &#xBCD1;&#xC6D0; &#xADE0;&#xC5D0; &#xB300; &#xD55C; &#xACE0;&#xC720;&#xD55C; &#xD638;&#xC2A4;&#xD2B8; &#xBC29;&#xC5B4; &#xC804;&#xB7B5;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xBE44;&#xC815;&#xD615; miRNA &#xD65C;&#xB3D9;&#xC744; &#xD1B5;&#xD574; &#xC77C;&#xBD80; &#xC140; &#xB0B4;&#xC7A5; &#xC800;&#xD56D; &#xC804;&#xC2DC;.

Plasmodium falciparum&#xC5D0; &#xACB8; &#xC0C1; &#xC801;&#xD608;&#xAD6C; &#xC801;&#xD608;&#xAD6C; Micrornas&#xC758; &#xC804; &#xAE30;&#xC0DD;&#xCDA9; &#xBC88;&#xC5ED; &#xC5B5;&#xC81C; &#xBC0F; &#xB9D0;&#xB77C;&#xB9AC;&#xC544; &#xC800;&#xD56D;&#xC5D0; &#xAE30;&#xC5EC; &#xD55C;&#xB2E4;.

Eritrocitos con un alelo de la variante de la hemoglobina (HbS), que causa la enfermedad de c&eacute;lulas falciformes y resiste la infecci&oacute;n por el par&aacute;sito de la malaria Plasmodium falciparum. La base molecular de esta resistencia, que ha sido reconocida como multifactorial, sigue siendo incompleto entendida. Aqu&iacute; mostramos que la composici&oacute;n dysregulated de microARN (miARN), heterocigoto HBA o homocig&oacute;ticos HbSS eritrocitos, contribuye a la resistencia contra p. falciparum. Durante el ciclo de vida de intraerythrocytic de p. falciparum, un subconjunto de eritrocito miRNAs translocan en el par&aacute;sito. Dos miRNAs, miR-451 y dej&oacute;-7i, eran altamente enriquecidos en eritrocitos HbAS y HbSS, y estos miRNAs, junto con el miR-223, hab&iacute;an regulada negativamente el crecimiento del par&aacute;sito. Sorprendentemente, encontramos que miR-451 y dej&oacute;-7i integrado en par&aacute;sito esencial mensajero ARN y, v&iacute;a carga ribosomal deteriorada, dio lugar a la inhibici&oacute;n traduccional. Por lo tanto, los eritrocitos falciformes exhiben resistencia celular intr&iacute;nseca a la malaria en parte a trav&eacute;s de una actividad de miRNA anormal, que puede representar una estrategia de defensa &uacute;nico anfitri&oacute;n frente a pat&oacute;genos eucariotas complejos.

Desplazamiento de c&eacute;lulas falciformes eritrocito microRNAs en Plasmodium falciparum inhibe traducci&oacute;n del par&aacute;sito y contribuye a la resistencia de la malaria.

Oncogenic mutations in the small Ras GTPases KRas, HRas, and NRas render the proteins constitutively GTP bound and active, a state that promotes cancer. Ras proteins share ~85% amino acid identity, are activated by and signal through the same proteins, and can exhibit functional redundancy. Nevertheless, manipulating expression or activation of each isoform yields different cellular responses and tumorigenic phenotypes, even when different ras genes are expressed from the same locus. We now report a novel regulatory mechanism hardwired into the very sequence of RAS genes that underlies how such similar proteins impact tumorigenesis differently. Specifically, despite their high sequence similarity, KRAS is poorly translated compared to HRAS due to enrichment in genomically underrepresented or rare codons. Converting rare to common codons increases KRas expression and tumorigenicity to mirror that of HRas. Furthermore, in a genome-wide survey, similar gene pairs with opposing codon bias were identified that not only manifest dichotomous protein expression but also are enriched in key signaling protein classes and pathways. Thus, synonymous nucleotide differences affecting codon usage account for differences between HRas and KRas expression and function and may represent a broader regulation strategy in cell signaling.

Rare codons regulate KRas oncogenesis.

Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.

Joshua Lacsina

Department of Cell Biology

Duke University School of Medicine

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Joshua Lacsina

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology

Duke University School of Medicine

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Department of Cell Biology