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1.
Proc Natl Acad Sci U S A ; 116(8): 2935-2944, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718402

RESUMO

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.


Assuntos
Proteínas ELAV/química , Proteína Semelhante a ELAV 1/química , Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato/genética , Cristalografia por Raios X , Dimerização , Proteína Semelhante a ELAV 1/genética , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Ligação a RNA/genética , Ribonucleosídeo Difosfato Redutase/química , Proteínas Supressoras de Tumor/química
2.
J Cell Biol ; 213(2): 173-84, 2016 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-27114500

RESUMO

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.


Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Pseudópodes/fisiologia , Transporte Biológico , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Exossomos/fisiologia , Exossomos/ultraestrutura , Células HEK293 , Humanos , Microscopia Eletrônica de Varredura , Pseudópodes/ultraestrutura
3.
Nanomedicine ; 11(4): 879-83, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25659648

RESUMO

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.


Assuntos
Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/ultraestrutura , Cromatografia em Gel , Células HEK293 , Humanos , Ultrafiltração
4.
EMBO J ; 32(8): 1115-27, 2013 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23511973

RESUMO

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.


Assuntos
Retículo Endoplasmático/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Argonautas/análise , RNA Helicases DEAD-box/análise , Retículo Endoplasmático/química , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Ligação a RNA/análise , Ribonuclease III/análise
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