A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene.

mdm2 encodes for an E3 ubiquitin ligase targeting constitutively expressed p53 for proteasomal degradation. Several protein isoforms have been described for human MDM2 (HDM2), some of which may correspond to splicing variants detectable by RT-PCR in many tumors. Upon cellular stress, p53 becomes resistant to MDM2 and, in a feedback loop, up-regulates mdm2 transcription. The physiological relevance of stress-induced mdm2 gene activity is not well understood. We describe a small nuclear RNA of 365 bases comprised of the first five hdm2 exons and lacking polyadenylation. hdm365 precedes full-length hdm2 RNA expression after induction by p53 and accumulates to significant levels in the nucleus, detectable at the site of hdm2 transcription and processing only. Considering a 10-fold lower stability and high steady-state levels of the novel RNA species, hdm365 appears to be the major processing product of hdm2 transcripts. hdm365 induction was observed after ectopic expression of p53 and after DNA damaging treatment of tumor cell lines, primary fibroblasts and lymphocytes, and was not related to apoptosis. Corresponding truncated transcripts were observed in hdm2 amplified cells. High stress-inducible expression levels, absence of a corresponding protein, and nuclear localisation of hdm365 suggest a novel RNA-based function for hdm2.

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome.

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.