Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro.

Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1α cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1α generated 2', 3'-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA.

Antifreeze activity of cold acclimated Japanese radish and purification of antifreeze peptide.

Japanese radish tuber and leaf produced antifreeze proteins (AFPs) having thermal hysteresis activity (TH) and ice recrystallization inhibiting activity (RI). Upon cold acclimation, the apoplastic fluid of the Japanese radish exhibited hexagonal crystal growth, indicating the presence of an antifreeze protein. The induction patterns of protein and the TH activity of apoplastic fraction from both samples were different. The TH activities of apoplastic fraction from tuber and leaves were 0.20 +/- 0.03 and 0.18 +/- 0.02 degree C, respectively. Also, the TH and RI activities of apoplastic fraction of leaves were activated by autoclave treatment at pH 10.0. An antifreeze peptide (molecular weight 1320), was purified using chromatography. Furthermore, the chitinase and beta-1, 3-glucanase activities in the apoplastic fraction of its tuber were induced by the cold acclimation. Some proteins in this apoplastic fraction were reacted with the anti-glucanase-like protein (GLP) antiserum and anti-chitinase-like protein (CLP) antiserum produced against isolated winter rye AFPs. This is the first report on the presence and characterization of AFPs from Japanese radish tuber.