Abstract
Stress granules (SG) and processing bodies (PBs) are cytoplasmic ribonucleoprotein particles whose assembly is induced by different stimuli. SG are the site of storage of untranslated transcripts formed in response to environmental stress, whereas PBs are involved in mRNA turnover. We recently characterized a novel family of four human proteins related to the Caenorhabditis elegans Mex-3, a RNA binding protein involved in the establishment of the anterior-posterior embryonic asymmetry and in the maintenance of germline pluripotency. We now report that the adaptor proteins 14-3-3 bind to hMex-3B but not to the three other hMex-3 family members. Serine 462, when phosphorylated, is the major 14-3-3 docking site on hMex-3B, and manipulation of this interaction reveals that 14-3-3 both stabilizes hMex-3B and modulates its ability to bind RNA. Furthermore, the complex formed between hMex-3B and Argonaute proteins is excluded from PBs when the interaction with 14-3-3 is disrupted, whereas the recruitment to SG is not affected. Thus, 14-3-3 exerts combined effects on hMex-3B and acts as a major regulator of the sorting between distinct classes of RNA granules.
Highlights
Transcripts [1,2,3,4]
A large body of work has demonstrated that Stress granules (SG) function as a triage center, redirecting mRNA fated to be degraded to processing bodies (PBs) or allowing their transport to the cytoplasm where translation is reinitiated on polysomes [10, 11]
We and others have recently identified a family of four human genes homologous to the Caenorhabditis elegans Mex-3 that codes a RNA-binding protein which contains two tandemly repeated heterogeneous nuclear ribonucleoprotein K homology (KH) domains [13, 14]
Summary
Constructs and Mutagenesis—Coding sequences for hMex3A, hMex-3B, hMex-3C, and p62-sequestosome were cloned in pCMV-Tag3B vector (Stratagene) as described previously [14]. Mouse monoclonal anti-14-3-3 (H-8) antibody (Santa Cruz Biotechnology) and anti-Phospho(Ser) 14-3-3 binding motif (4E2) antibody (Cell Signaling) were used in Western blots at 1:1000 dilution. HeLa cells were grown in Dulbecco’s modified Eagle’s medium with 1 g/ liter glucose supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Protein Analyses and Immunoprecipitation—Cells were lysed 48 h post-transfection, and Western blot analyses and immunoprecipitations were performed as described earlier [14, 23] (see supplemental “Materials and Methods”). Quantitative Western blot analyses were performed using Li-Cor Odissey (Li-Cor Biosciences) according to the manufacturer’s instruction (see supplemental “Materials and Methods”). For characterization and quantification of hMex-3B containing granules, cells were transfected and stained as described above, and protein localization was observed by confocal microscopy. Signal intensity was quantified using a FluorS Multimager and QuantityOne (Bio-Rad)
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