Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.Nonsense-mediated mRNA decay (NMD) rapidly and selectively degrades eukaryotic mRNAs containing premature translation termination codons (PTCs) (reviewed in references 27, 43, and 65). NMD likely improves the fidelity of gene expression by degrading aberrant mRNAs, thereby protecting cells from potentially deleterious consequences of their translation (9, 28, 53). The biological sources of NMD substrates are only partially understood. For example, unspliced, unproductively spliced, and aberrantly spliced mRNAs are degraded by NMD (29,39,48). NMD strongly influences the expression of certain genes for which mRNAs containing PTCs are a normal feature of their expression. For example, gene rearrangements of T-cell receptor genes often result in mRNAs that contain PTCs and are subjected to NMD (11).Gene products required for NMD have been identified in fungi, nematodes, insects, and mammals (13,16,24,49), and putative orthologs of these genes are evident in many sequenced eukaryotic genomes. A core group of three genes first identified in the yeast Saccharomyces cerevisiae (UPF1, NMD2/ UPF2, and UPF3) (reviewed in reference 69) are required for NMD in all tested eukaryotes (3,40,45,50,57,61). The conservation of both the sequence and function of such genes indicates that NMD is an evolutionarily ancient process and suggests that elements of the mechanism of NMD will be similar in most, or perhaps all, eukaryotes.PTCs are distinguished from normal termination codons in both yeast and mammalian cells by the context of translation termination. Specific cis-acting elements mark the open reading frame of mRNAs and activate NMD when translation terminates upstream of the marks. The Upf proteins appear to be involved both in sensing the presence of downstream signals and in activating the degradation machinery. In yeast cells, the cis-acting elements are termed downstream sequence elements, which are thought to be present throughout coding sequences but not in 3Ј untranslated regions (54). Hrp1p binds both downstream seq...

Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed "nonsense-mediated mRNA decay" (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with ("marks") those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.

We describe a platform that utilizes wheat germ cell‐free technology to produce protein samples for NMR structure determinations. In the first stage, cloned DNA molecules coding for proteins of interest are transcribed and translated on a small scale (25 µL) to determine levels of protein expression and solubility. The amount of protein produced (typically 2–10 µg) is sufficient to be visualized by polyacrylamide gel electrophoresis. The fraction of soluble protein is estimated by comparing gel scans of total protein and soluble protein. Targets that pass this first screen by exhibiting high protein production and solubility move to the second stage. In the second stage, the DNA is transcribed on a larger scale, and labeled proteins are produced by incorporation of [15N]‐labeled amino acids in a 4 mL translation reaction that typically produces 1–3 mg of protein. The [15N]‐labeled proteins are screened by 1H‐15N correlated NMR spectroscopy to determine whether the protein is a good candidate for solution structure determination. Targets that pass this second screen are then translated in a medium containing amino acids doubly labeled with 15N and 13C. We describe the automation of these steps and their application to targets chosen from a variety of eukaryotic genomes: Arabidopsis thaliana, human, mouse, rat, and zebrafish. We present protein yields and costs and compare the wheat germ cell‐free approach with alternative methods. Finally, we discuss remaining bottlenecks and approaches to their solution.

The Center for Eukaryotic Structural Genomics, in cooperation with Ehime University and CellFree Sciences, has developed a novel wheat germ cell-free technology for the production of eukaryotic proteins. Protein production and purification are robust and scalable for high-throughput applications. The protocols have been used to express and purify proteins from Arabidopsis thaliana, human, mouse, rat and zebra fish. This unit describes expression and purification protocols for both small-scale testing (microgram) and large-scale production (milligram) of N-His6- and N-GST-tagged proteins. The methods described in this unit can be used to produce both unlabeled and labeled proteins required for structure-based determinations by NMR spectroscopy or X-ray crystallography.

The sorting nexins (SNXs) constitute a large group of PX domain-containing proteins that play critical roles in protein trafficking. We report here the solution structure of human sorting nexin 22 (SNX22). Although SNX22 has <30% sequence identity with any PX domain protein of known structure, it was found to contain the a/b fold and compact structural core characteristic of PX domains. Analysis of the backbone dynamics of SNX22 by NMR relaxation measurements revealed that the two walls of the ligand binding cleft undergo internal motions: on the picosecond timescale for the b1/b2 loop and on the micro-to millisecond timescale for the loop between the polyproline motif and helix a2. Regions of the SNX22 structure that differ from those of other PX domains include the loop connecting strands b1 and b2 and the loop connecting helices a1 and a2, which appear to be more mobile than corresponding loops in other known structures. The interaction of dibutanoyl-phosphatidylinositol-3-phosphate (dibutanoyl-PtdIns(3)P) with SNX22 was investigated by an NMR titration experiment, which identified the binding site in a basic cleft and indicated that ligand binding leads only to a local structural rearrangement as has been found with other PX domains. Because motions in the loops are damped out when dibutanoyl-PtdIns(3)P binds, entropic effects could contribute to the lower affinity of SNX22 for this ligand compared to other PX domains.