Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed ''SURF'' that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.[Keywords: NMD; mRNA surveillance; UPF1; SMG-1; PIKK; translation termination; mRNP remodeling] Supplemental material is available at http://www.genesdev.org.

The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/ NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

DREF, a transcription regulatory factor which specifically binds to the promoter-activating element DRE (DNA replication-related element) of DNA replicationrelated genes, was purified to homogeneity from nuclear extracts of Drosophila Kc cells. cDNA for DREF was isolated with the reverse-transcriptase polymerase chain reaction method using primers synthesized on the basis of partial amino acid sequences and following screening of cDNA libraries. Deduced from the nucleotide sequences of cDNA, DREF is a polypeptide of 701 amino acid residues with a molecular weight of 80,096, which contains three characteristic regions, rich in basic amino acids, proline, and acidic amino acids, respectively. Deletion analysis of bacterially expressed DREF fused with glutathione S-transferase (GST-DREF) indicated that a part of the N-terminal basic amino acid region (16 -115 amino acids) is responsible for the specific binding to DRE. A polyclonal and four monoclonal antibodies were raised against the GST-DREF fusion protein. The antibodies inhibited specifically the transcription of DNA polymerase ␣ promoter in vitro. Cotransfection experiments using Kc cells demonstrated that overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknü llt gene product. These results confirmed that DREF is a trans-activating factor for DNA replication-related genes. Immunocytochemical analysis demonstrated the presence of DREF polypeptide in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences.

The results of these novel ELISAs indicated that IgG anti-Dsc autoantibodies were frequently detected and potentially pathogenic in nonclassical pemphigus.