purification-microrna-profiling-exosomes-derived-from-blood-culture

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Drexel University College of Medicine

RNA containing 5-fluorouridine (F(5)U) had previously been used to examine the mechanism of the pseudouridine synthase TruA, formerly known as pseudouridine synthase I [Gu et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 14270-14275]. From that work, it was reasonably concluded that the pseudouridine synthases proceed via a mechanism involving a Michael addition by an active site aspartic acid residue to the pyrimidine ring of uridine or F(5)U. Those conclusions rested on the assumption that the hydrate of F(5)U was obtained after digestion of the product RNA and that hydration resulted from hydrolysis of the ester intermediate between the aspartic acid residue and F(5)U. As reported here, (18)O labeling definitively demonstrates that ester hydrolysis does not give rise to the observed hydrated product and that digestion generates not the expected mononucleoside product but rather a dinucleotide between a hydrated isomer of F(5)U and the following nucleoside in RNA. The discovery that digestion products are dinucleotides accounts for the previously puzzling differences in the isolated products obtained following the action of the pseudouridine synthases TruB and RluA on F(5)U in RNA.

The handling of the mechanistic probe 5-fluorouridine by the pseudouridine synthase TruA and its consistency with the handling of the same probe by the pseudouridine synthases TruB and RluA.

ARN contenant 5-fluorouridine (F(5)U) avait d&eacute;j&agrave; &eacute;t&eacute; utilis&eacute; pour examiner le m&eacute;canisme de la pseudouridine synthase TruA, anciennement appel&eacute;e pseudouridine synthase I [Proc Natl. Acad. Sci. U.S.A. 96, 14270-14275 de Gu et coll. (1999)]. De ce travail, il &eacute;tait raisonnable de conclure que les pseudouridine synthases continuer via un m&eacute;canisme impliquant une addition de Michael par un r&eacute;sidu d&#39;acide aspartique de site actif pour le cycle pyrimidine de l&#39;uridine ou U. F (5) Ces conclusions reposaient sur l&#39;hypoth&egrave;se que l&#39;hydrate de F (5) U a &eacute;t&eacute; obtenu apr&egrave;s la digestion du produit RNA et cette hydratation r&eacute;sultant de l&#39;hydrolyse de l&#39;ester interm&eacute;diaire entre le r&eacute;sidu d&#39;acide aspartique et U. F (5) Comme indiqu&eacute; ici, (18) O &eacute;tiquetage d&eacute;finitivement d&eacute;montre que hydrolyse de l&#39;ester ne donne pas lieu au produit hydrat&eacute; observ&eacute; et que la digestion g&eacute;n&egrave;re pas le produit de mononucleoside attendu, mais plut&ocirc;t un dinucl&eacute;otide entre un isom&egrave;re hydrat&eacute; de F (5) vous et le nucl&eacute;oside suivant dans l&#39;ARN. La d&eacute;couverte que les produits de la digestion sont dinucl&eacute;otides prend en compte les diff&eacute;rences d&eacute;j&agrave; d&eacute;routant dans les produits isol&eacute;s obtenus suite &agrave; l&#39;action des synthases pseudouridine TruB et RluA sur F (5) U dans l&#39;ARN.

La manipulation de la sonde m&eacute;canistique 5-fluorouridine par la pseudouridine synthase TruA et sa coh&eacute;rence avec la manipulation de la sonde m&ecirc;me par les synthases pseudouridine TruB et RluA.

RNA contenente 5-fluorouridina (F(5)U) erano stati precedentemente utilizzati per esaminare il meccanismo del pseudouridine synthase Tr&#x1B0;a, precedentemente noto come pseudouridine synthase I [Gu et al (1999) proc Acad Sci. U.S.A. 96, 14270-14275 Natl.]. Da quel lavoro, si &egrave; ragionevolmente concluso che la synthases pseudouridine procedere attraverso un meccanismo che coinvolge un aggiunta di Michael da un residuo di acido aspartico sito attivo per l&#39;anello pirimidinico di uridina o U. F (5) Tali conclusioni si riposava sul presupposto che l&#39;idrato di F (5) U &egrave; stata ottenuta dopo digestione del prodotto RNA e che idratazione derivato dall&#39;idrolisi dell&#39;estere intermedio tra il residuo di acido aspartico e la U. F (5) Come segnalato qui, (18) O etichettatura definitivamente dimostra che idrolisi dell&#39;estere non danno luogo al prodotto osservato idratato e che digestione genera non previsto mononucleoside prodotto, ma piuttosto un dinucleotide tra un isomero idratato di F (5) si e il nucleoside seguente nel RNA. La scoperta che i prodotti di digestione sono dinucleotidi conti per le differenze in precedenza sconcertante in isolati prodotti ottenuti seguendo l&#39;azione della synthases pseudouridine TruB e RluA su F (5) U nel RNA.

La gestione della sua coerenza e la sonda meccanicistica 5-fluorouridina dal pseudouridine synthase Tr&#x1B0;a con la manipolazione della sonda stessa dalla synthases pseudouridine TruB e RluA.

Subcellular localization of mRNA enables compartmentalized regulation within large cells. Neurons are the longest known cells; however, so far, evidence is lacking for an essential role of endogenous mRNA localization in axons. Localized upregulation of Importin Î²1 in lesioned axons coordinates a retrograde injury-signaling complex transported to the neuronal cell body. Here we show that a long 3' untranslated region (3' UTR) directs axonal localization of Importin Î²1. Conditional targeting of this 3' UTR region in mice causes subcellular loss of Importin Î²1 mRNA and protein in axons, without affecting cell body levels or nuclear functions in sensory neurons. Strikingly, axonal knockout of Importin Î²1 attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. Thus, localized translation of Importin Î²1 mRNA enables separation of cytoplasmic and nuclear transport functions of importins and is required for efficient retrograde signaling in injured axons.

Subcellular knockout of importin Î²1 perturbs axonal retrograde signaling.

Localisation subcellulaire des ARNm permet compartiment&eacute;e r&egrave;glement au sein de grandes cellules. Les neurones sont les cellules les plus longues connues ; Toutefois, jusqu&#39;&agrave; pr&eacute;sent, preuves manquent pour un r&ocirc;le essentiel de localisation d&#39;ARNm endog&egrave;ne dans les axones. Localis&eacute;e upregulation of Importin &beta;1 dans les axones l&eacute;s&eacute;s coordonne un complexe blessure-signalisation r&eacute;trograde transport&eacute; vers le corps cellulaire neuronal. Ici, nous montrons qu&#39;une r&eacute;gion longue de 3&#39; non traduite (3&#39; UTR) ordonne &agrave; localisation axonale d&#39;important &beta;1. Conditionnelle de ciblage de ce 3&#39; r&eacute;gion UTR chez la souris provoque perte subcellulaire de Importin &beta;1 ARNm et de prot&eacute;ines dans les axones, sans affecter les niveaux du corps cellulaire ou des fonctions nucl&eacute;aires dans les neurones sensoriels. &Eacute;tonnamment, knockout axonal &beta;1 Importin att&eacute;nue les r&eacute;ponses transcriptionnelles corps cellulaire pour nerveuse des blessures et la r&eacute;cup&eacute;ration fonctionnelle in vivo des retards. Ainsi, une traduction localis&eacute;e de &beta;1 Importin mRNA favorise la s&eacute;paration des fonctions de transport cytoplasmiques et nucl&eacute;aires d&#39;importines et est requis pour la signalisation r&eacute;trograde efficace dans bless&eacute; axones.

Knockout subcellulaire d&#39;important &beta;1 perturbe axonal r&eacute;trograde de la signalisation.

Localizzazione sottocellulare di mRNA consente compartimenti regolamento all&#39;interno delle cellule di grandi dimensioni. I neuroni sono le cellule pi&ugrave; lunga conosciute; Tuttavia, finora, prove mancano per un ruolo essenziale di localizzazione di mRNA endogeno in assoni. Localizzata sovraregolazione di Importin &beta;1 in assoni elettivamente coordina un complesso di lesioni-segnalazione retrogrado trasportato al corpo delle cellule neuronale. Qui vi mostriamo che una regione lunga 3&#39; non tradotte (3&#39; UTR) dirige assonale localizzazione di Importin &beta;1. Condizionale di targeting di questa 3&#39; regione UTR nei topi provoca perdita sottocellulare di Importin &beta;1 mRNA e proteina in assoni, senza intaccare i livelli del corpo cellulare o funzioni nucleari nei neuroni sensoriali. Sorprendentemente, knockout assonale di Importin &beta;1 attenua risposte di transcriptional corpo cellulare per infortunio e ritardi recupero funzionale in vivo del nervo. Dunque, localizzate traduzione di &beta;1 Importin mRNA consente la separazione delle funzioni di trasporto nucleare e citoplasmatica di importins ed &egrave; necessaria per la segnalazione retrograda efficiente feriti assoni.

Article	Year
The handling of the mechanistic probe 5-fluorouridine by the pseudouridine synthase TruA and its consistency with the handling of the same probe by the pseudouridine synthases TruB and RluA. Biochemistry\| PubMed ID: 21142053	2011
Subcellular knockout of importin Î²1 perturbs axonal retrograde signaling. Neuron\| PubMed ID: 22841314	2012

Marguerite K. McDonald

Biography

JoVE Journal Publications

Other Publications