Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Vibrio vulnificus is a halophilic estuarine bacterium which causes fatal septicemia and necrotizing wound infections, especially in patients with hepatic disease, heavy alcohol drinking habits and hemochromatosis. V. vulnificus septicemia is characterized by rapid and fulminant progression, and results in a high mortality rate of over 50%. 1) Several bacterial components have been suggested to be virulence factors of V. vulnificus.2,3) Of these, an extracellular hemolysin or cytolysin (VvhA) and an extracellular protease (VvpE) have been the most extensively studied factors. VvhA, the most potent exotoxin, kills mice and shows a variety of biological activities including hemolysis or cytolysis, apoptosis, vasodilatation, and so on. [4][5][6][7][8] In animal studies, the injection of purified VvhA reproduces the same pathological manifestations of septicemia as caused by the injection of live bacteria. 4,9,10) VvpE also exhibits a host of biological activities including dermonecrosis, edema, and ulceration, and increased vascular permeability. [11][12][13][14] However, the pathogenetic significance of both VvhA and VvpE has been brought into serious doubt by mouse-lethality studies of VvhA-and/or VvpE-deficient mutants. [15][16][17] The inactivation of vvhA gene does not affect the mouse-lethality. This raises the possibility that only very small amounts of VvhA may be produced in vivo, 18,19) and the produced VvhA may be rapidly inactivated by host factors such as cholesterol and bacterial factors such as VvpE. 5,16,[20][21][22] Therefore, in order to evidently determine the pathogenetic roles of VvhA, further detailed studies regarding the in vitro and in vivo production and inactivation of VvhA are necessary.Physiologically, VvhA is produced in the early growth phase, and becomes abruptly inactivated in the late growth phase with the concomitant production of VvpE. Accordingly, it has been classically believed that the inactivation of VvhA is attributable to the destruction of VvhA by VvpE. 9)Recently, it has also been reported that the activity of VvhA in the culture supernatant of a VvpE-deficient mutant was twice that of the wild-type strain, and persisted for a much longer period, suggesting that VvhA might be a substrate of VvpE.16) However, direct evidence for the destruction of VvhA by VvpE has never been presented. From the standpoint of evolution, some doubt also exists as to whether VvhA is destroyed by VvpE or other proteases. In addition, if VvpE or other proteases can destroy and inactivate VvhA, the routine functional assay measuring hemolytic activity using red blood cells (RBC) may not reflect the actual production of VvhA. In this study, therefore, we attempted to obtain direct evidence for the inactivation of VvhA in the late growth phase. Surprisingly, we observed that the inactivation of VvhA was due to the novel oligomerization of VvhA by unknown mechanism, but not to the destruction of VvhA by VvpE. MATERIALS AND METHODSMedia, Bacterial Strains, Plasmids, and Primers 2.5% NaCl-Heart Infus...

Abstract. Osteocalcin expression is restricted to osteoblasts and serum osteocalcin level is elevated in metastatic bone tumors including prostate tumors, which predominantly metastasizes to the bone and causes typical osteoblastic lesions. Previously, we have reported that osteocalcin RNA is widely expressed but incompletely spliced in the prostate including prostate tumors. Considering that many studies using osteocalcin-driven gene therapy have been conducted to treat hormone refractory metastatic tumors, detailed mechanisms controlling osteocalcin expression needs to be clarified. We aim to learn how osteocalcin expression is regulated during the metastatic process of prostate cancer. We applied assays of immunohistochemistry and RNA in situ hybridization in prostate tumors acquired from prostate (15) and metastatic sites, 13 from lymph node and 14 from bone. RT-PCR analysis in various cultured prostate cells was also performed. As predicted, osteocalcin RNA was highly expressed in most prostate epithelial cells of tumors, regardless of metastatic status of the tumor. However, osteocalcin protein was undetectable in tumors acquired from the primary site or lymph nodes whereas protein was highly expressed in the majority of bone-metastasized prostate tumors. RT-PCR analysis demonstrated that there was more completely spliced form of osteocalcin RNA present in bone-derived prostate cancer cells. Our data suggest that osteocalcin RNA was expressed but not completely spliced in non-bone environment, ultimately resulting in improper production of osteocalcin protein. This study explains why serum osteocalcin level is increased in patients with bone-metastasized prostate cancers. Yet, it remains to be clarified what regulates bone-specific osteocalcin RNA splicing in prostate tumors.

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3′UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3′UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3.