Upf1 and Upf2 proteins mediate normal yeast mRNA degradation when translation initiation is limited.

mRNA degradation is coupled with the process of mRNA translation. For example, an mRNA molecule, on which translation is prematurely terminated because of a nonsense codon, may be rapidly degraded. This nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae is mediated by the Upf1 and Upf2 proteins. Yeast mRNAs can also be selectively destabilized by limiting the rate of translation initiation. Two such destabilized mRNAs, from the SSA1 and SSA2 genes, have been identified using temperature-sensitive mutations affecting the Prt1 component of eukaryotic initiation factor 3. For SSA1 and SSA2 mRNAs, and for structurally modified SSA mRNA derivatives, I show here that degradation is triggered when translation initiation is limited but ongoing. This initiation-dependent mRNA degradation is limited to a subset of mRNAs that includes at least those from the SSA1 and SSA2 genes, and occurs through Upf1- and Upf2-mediated processes, although sequence elements characteristic of nonsense-mediated decay are not evident in these mRNAs.     

A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA.

In Saccharomyces cerevisiae, seven snRNAs (snR3, 4, 5, 8, 9, 10 and 17) are retained in the nucleus under conditions in which nucleoplasmic RNAs are lost, and may be nucleolar. All of these snRNAs show properties consistent with hydrogen bonding to pre-ribosomal RNAs; snR5 and 8 with 20S pre-rRNA, snR3, 4, 10 and 17 with 35S pre-rRNA and snR9 with 20-35S RNA. Strains lacking snR10 are impaired in growth and specifically defective in the processing of 35S RNA. Processing is slowed, leading to 35S RNA accumulation and most cleavage occurs, not at the normal sites, but at sites which in wild-type strains are used for subsequent steps in rRNA maturation.     

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.     

Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis.

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GAL10 promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOP1 and GAR1 and in an snr10 mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and B1 and not, as previously suggested, at the B1 site, snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.     

Retrieval of HDEL proteins is required for growth of yeast cells

The ERD2 gene of Saccharomyces cerevisiae encodes the receptor which retrieves HDEL-containing containing ER proteins from the Golgi apparatus. Viable erd2 mutants have been isolated that show no obvious HDEL-dependent retention of the luminal ER protein BiP, suggesting that retrieval of HDEL proteins is not essential for growth. However, cells that lack Erd2p completely have a defective Golgi apparatus and cannot grow. This observation led to the suggestion that the receptor had a second function, possibly related to its ability to recycle from Golgi to ER. In this paper we investigate the requirements for Erd2p to support growth. We show that mutations that block its recycling also prevent growth. In addition, we show that all mutant receptors that can support growth have a residual ability to retrieve BiP, which is detectable when they are overexpressed. Mere recycling of an inactive form of the receptor, mediated by a cytoplasmic KKXX sequence, is not sufficient for growth. Furthermore, saturation of the receptor by expression of an HDEL-tagged version of pro-alpha factor inhibits growth, even of strains that do not show obvious BiP retention. We conclude that growth requires the HDEL-dependent retrieval of one or more proteins, and that these proteins can be recognized even under conditions where BiP is secreted. Genetic screens have failed to identify any one protein whose loss could account for the Erd2p requirement. Therefore, a growth may require the retention of multiple HDEL proteins in the ER, or alternatively the removal of such proteins from the Golgi apparatus.     

Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast.

Rapid turnover of nonsense-containing mRNAs in the yeast Saccharomyces cerevisiae is dependent on the products of the UPF1 (Upf1p), NMD2/UPF2 (Nmd2p) and UPF3 (Upf3p) genes. Mutations in each of these genes lead to the selective stabilization of mRNAs containing early nonsense mutations without affecting the decay rates of most other mRNAs. NMD2 was recently identified in a two-hybrid screen as a gene that encodes a Upf1p-interacting protein. To identify the amino acids essential to this interaction, we used two-hybrid analysis as well as missense, nonsense, and deletion mutants of NMD2, and mapped the Upf1p-interacting domain of Nmd2p to a 157-amino acid segment at its C-terminus. Mutations in this domain that disrupt interaction with Upf1p also disrupt nonsense-mediated mRNA decay. A dominant-negative deletion allele of NMD2 identified previously includes the Upf1p-interacting domain. However, mutations in the Upf1p-interacting domain do not affect dominant-negative inhibition of mRNA decay caused by this allele, suggesting interaction with yet another factor. These results, and the observation that deletion of a putative nuclear localization signal and a putative transmembrane domain also inactivate nonsense-mediated mRNA decay, suggest that Nmd2p may contain as many as four important functional domains.     

Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif

Clathrin-coated vesicles (CCVs) are a central component of endocytosis and traffic between the trans-Golgi network (TGN) and endosomes. Although endocytic CCV formation is well characterized, much less is known about CCV formation at internal membranes. Here we describe two epsin amino-terminal homology (ENTH) domain-containing proteins, Ent3p and Ent5p, that are intimately involved in clathrin function at the Golgi. Both proteins associate with the clathrin adaptor Gga2p in vivo; Ent5p also interacts with the clathrin adaptor complex AP-1 and clathrin. A novel, conserved motif that mediates the interaction of Ent3p and Ent5p with gamma-ear domains of Gga2p and AP-1 is defined. Ent3p and Ent5p colocalize with clathrin, and cells lacking both Ent proteins exhibit defects in clathrin localization and traffic between the Golgi and endosomes. The findings suggest that Ent3p and Ent5p constitute a functionally related pair that co-operate with Gga proteins and AP-1 to recruit clathrin and promote formation of clathrin coats at the Golgi/endosomes. On the basis of our results and the established roles of epsin and epsin-related proteins in endocytosis, we propose that ENTH-domain-containing proteins are a universal component of CCV formation.     

Intra- and Intermolecular Regulatory Interactions in Upf1, the RNA Helicase Central to Nonsense-Mediated mRNA Decay in Yeast

RNA helicases are involved in almost every aspect of RNA metabolism, yet very little is known about the regulation of this class of enzymes. In Saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mRNAs are controlled by the group I RNA helicase Upf1 and the proteins it interacts with, Upf2 and Upf3. Combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine- and histidine-rich (CH) domain and the RNA helicase domain of yeast Upf1 can engage in two new types of molecular interactions: an intramolecular interaction between these two domains and self-association of each of these domains. Multiple observations indicate that these molecular interactions are crucial for Upf1 regulation. First, coexpression of the CH domain and the RNA helicase domain in trans can reconstitute Upf1 function in both promoting nonsense-mediated mRNA decay (NMD) and preventing nonsense suppression. Second, mutations that disrupt Upf1 intramolecular interaction cause loss of Upf1 function. These mutations weaken Upf2 interaction and, surprisingly, promote Upf1 self-association. Third, the genetic defects resulting from deficiency in Upf1 intramolecular interaction or RNA binding are suppressed by expression of Upf2. Collectively, these data reveal a set of sequential molecular interactions and their roles in regulating Upf1 function during activation of NMD and suggest that cis intramolecular interaction and trans self-association may be general mechanisms for regulation of RNA helicase functions.     

Sphingolipids are required for the stable membrane association of glycosylphosphatidylinositol-anchored proteins in yeast

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.     

The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its