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Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRESdependent translation, indicating a novel function for the cytoplasmic form of this protein.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5 untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.

A poptosis, or programmed cell death, is important for the normal development of organisms and maintaining tissue homeostasis. Misregulation of apoptosis may lead to neurodegenerative disorders when apoptosis is increased and tumor formation when apoptosis is inhibited; thus, the pathway is tightly controlled, with both pro-and antiapoptotic factors playing a role. Of particular interest are members of the inhibitor of apoptosis (IAP) and Bcl-2 families of proteins that intercept virtually all apoptotic signals in the cell.X chromosome-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family; it directly interacts with and inhibits caspases 3, 7, and 9 and is therefore a key regulator of apoptosis (20). In contrast, Bcl-x L controls apoptosis by maintaining mitochondrial membrane homeostasis (14). Interestingly, both the XIAP and Bcl-x L mRNAs contain an internal ribosome entry site (IRES) that allows them to be translated during cellular stress by a cap-independent mechanism when cap-dependent translation is inhibited, which is necessary for their protective roles in the cell (4,11,16,24,49). Under normal growth conditions, translation of cellular mRNAs occurs through a cap-dependent mechanism that requires interaction of specific initiation factors (such as eukaryotic initiation factor 4E [eIF4E]) with the 5= cap of the mRNA, followed by recruitment of ribosomal subunits, recognition of the AUG start codon, and commencement of polypeptide chain elongation (reviewed in reference 26). However, certain cellular stresses such as nutrient deprivation, hypoxia, or low-dose irradiation cause attenuation of cap-dependent translation, and yet, under these conditions, a sizeable proportion of cellular mRNAs, perhaps as much as 10%, have been shown to be translated by a cap-independent mechanism, such as through an IRES (23,28,39). IRES elements are located within the 5= untranslated region (UTR) of some cellular mRNAs and are believed to recruit the ribosome directly, thereby bypassing the requirement for the mRNA 5= cap and eIF4E. Moreover, while IRES-dependent translation requires some canonical translation initiation factors, most (if not all) cellular IRES elements require the activity of auxiliary RNA binding proteins that function as IRES trans-acting factors (ITAFs) (reviewed in references 28 and 32). The mechanism by which ITAFs function is poorly understood, and since different IRESs require different sets of ITAFs, elucidating the identity of all known ITAFs has proven challenging. Furthermore, it has been shown that ITAFs can function as either positive or negative regulators of IRES-mediated translation. For example, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was shown to act as a repressor of XIAP IRES-mediated translation (33), whereas La autoantigen (19), hnRNPC1 and hnRNPC2 (18), and HuR (11) have all been shown to have a stimulating effect on XIAP IRES-mediated translation.We have previously demonstrated that treatment of small-cell lung cancer cells with fibroblast gr...

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DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner.

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