The enzymes and control of eukaryotic mRNA turnover.

The degradation of eukaryotic mRNAs plays important roles in the modulation of gene expression, quality control of mRNA biogenesis and antiviral defenses. In the past five years, many of the enzymes involved in this process have been identified and mechanisms that modulate their activities have begun to be identified. In this review, we describe the enzymes of mRNA degradation and their properties.

Computational modeling and experimental analysis of nonsense-mediated decay in yeast.

A conserved mRNA surveillance system, referred to as nonsense-mediated decay (NMD), exists in eukaryotic cells to degrade mRNAs containing nonsense codons. This process is important in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins. Using computational modeling and experimental analysis, we define the alterations in mRNA turnover triggered by NMD in yeast.

The enhancer of decapping proteins, Edc1p and Edc2p, bind RNA and stimulate the activity of the decapping enzyme.

A major pathway of eukaryotic mRNA turnover initiates with deadenylation, which allows a decapping reaction leading to 5'-3' exonucleolytic degradation. A key control point in this pathway is the decapping of the mRNA. Two proteins, Edc1 and Edc2, were genetically identified previously as enhancers of the decapping reaction.

"Stemness": transcriptional profiling of embryonic and adult stem cells.

The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells, and several of these genes are clustered in the genome. When compared to differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown.

Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae.

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis.

Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes.

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy.

Crystal structure of the human natural killer cell inhibitory receptor KIR2DL1-HLA-Cw4 complex.

Inhibitory natural killer (NK) cell receptors down-regulate the cytotoxicity of NK cells upon recognition of specific class I major histocompatibility complex (MHC) molecules on target cells. We report here the crystal structure of the inhibitory human killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1) bound to its class I MHC ligand, HLA-Cw4. The KIR2DL1-HLA-Cw4 interface exhibits charge and shape complementarity.

Two related proteins, Edc1p and Edc2p, stimulate mRNA decapping in Saccharomyces cerevisiae.

The major mRNA decay pathway in Saccharomyces cerevisiae occurs through deadenylation, decapping, and 5' to 3' degradation of the mRNA. Decapping is a critical control point in this decay pathway. Two proteins, Dcp1p and Dcp2p, are required for mRNA decapping in vivo and for the production of active decapping enzyme.

The Puf3 protein is a transcript-specific regulator of mRNA degradation in yeast.

Eukaryotic post-transcriptional regulation is often specified by control elements within mRNA 3'- untranslated regions (3'-UTRs). In order to identify proteins that regulate specific mRNA decay rates in Saccharomyces cerevisae, we analyzed the role of five members of the Puf family present in the yeast genome (referred to as JSN1/PUF1, PUF2, PUF3, PUF4 and MPT5/PUF5). Yeast strains lacking all five Puf proteins showed differential expression of numerous yeast mRNAs.

Regulation of pancreas development by hedgehog signaling.

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue.

Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA.

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA.

A disulfide-linked natural killer cell receptor dimer has higher affinity for HLA-C than wild-type monomer.

Inhibitory receptors on the surface of natural killer (NK) cells recognize specific MHC class I molecules on target cells and prevent the target cell lysis by NK cells. The killer cell immunoglobulin-related receptors (KIR), KIR2D, found in human, specifically interact with polymorphic HLA-C molecules. The crystal structure of the inhibitory receptor, KIR2DL1, revealed a relationship to the hematopoietic receptor family, suggesting that the signaling mechanism of KIR2D molecules may resemble that of the hematopoietic receptors, and involve KIR2D dimerization.

Mechanisms and control of mRNA decapping in Saccharomyces cerevisiae.

The process of mRNA turnover is a critical component of the regulation of gene expression. In the past few years a discrete set of pathways for the degradation of polyadenylated mRNAs in eukaryotic cells have been described. A major pathway of mRNA degradation in yeast occurs by deadenylation of the mRNA, which leads to a decapping reaction, thereby exposing the mRNA to rapid 5' to 3' exonucleolytic degradation.

The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site.

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 A) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue.

Cobalt-mediated dimerization of the human natural killer cell inhibitory receptor.

Upon engagement of specific class I major histocompatibility complex (MHC) molecules on target cells, inhibitory receptors on natural killer (NK) cells deliver a negative signal that prevents the target cell lysis by NK cells. In humans, killer cell immunoglobulin-related receptors (KIR) with two immunoglobulin-like domains (KIR2D) modulate the lysis of target cells bearing specific HLA-C alleles (Moretta, A., Vitale, M.

Yeast Sm-like proteins function in mRNA decapping and decay.

One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5'-to-3' decay. A family of Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm' sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins (Lsm1-Lsm7) also lead to inhibition of mRNA decapping.

Aberrant mRNAs with extended 3' UTRs are substrates for rapid degradation by mRNA surveillance.

The mRNA surveillance system is known to rapidly degrade aberrant mRNAs that contain premature termination codons in a process referred to as nonsense-mediated decay. A second class of aberrant mRNAs are those wherein the 3' UTR is abnormally extended due to a mutation in the polyadenylation site. We provide several observations that these abnormally 3'-extended mRNAs are degraded by the same machinery that degrades mRNAs with premature nonsense codons.

The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.

The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5' to 3' exonucleolytic decay of the transcript body. To identify proteins that control the activity of the decapping enzyme, which is encoded by the DCP1 gene, we isolated a high-copy suppressor of the temperature-sensitive dcp1-2 allele, termed DCP2. Overexpression of Dcp2p partially suppressed the dcp1-2 decapping defect.

Screening for novel pancreatic genes expressed during embryogenesis.

We have combined suppressive subtractive hybridization with in situ hybridization to identify genes expressed at early stages of pancreas development. By using polymerase chain reaction amplification and subtractive hybridization, this protocol for screening can be applied when the amount of RNA is limited. Seven genes expressed in or adjacent to the pancreas anlage were isolated, three of which show similarity to known genes.

Analysis of mRNA decay pathways in Saccharomyces cerevisiae.

The analysis of mRNA turnover often requires a knowledge of the pathway by which a particular mRNA is being degraded. In this article we describe experimental procedures that can be used to determine the mechanism of degradation for yeast transcripts. These approaches include the insertion of strong secondary structures to block exonuclease cleavage and thereby trap decay intermediates.

Notochord repression of endodermal Sonic hedgehog permits pancreas development.

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-betaB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin.

Isolation and characterization of Dcp1p, the yeast mRNA decapping enzyme.

A major mechanism of mRNA decay occurs by the process of deadenylation, decapping and 5' --> 3' exonucleolytic degradation. Recently, the product of the DCP1 gene has been shown to be required for decapping mRNAs in vivo and co-purifies with decapping activity in vitro. We have purified Dcp1p to homogeneity and shown that it is sufficient for decapping, thereby indicating that Dcp1p is the decapping enzyme.

Analysis of the yeast genome: identification of new non-coding and small ORF-containing RNAs.

The genome sequences from increasing numbers of organisms allow for rapid and organized examination of gene expression. Yet current computational-based paradigms for gene recognition are limited and likely to miss genes expressing non-coding RNAs or mRNAs with small open reading frames (ORFs). We have utilized two strategies to determine if there are additional transcripts in the yeast Saccharomyces cerevisiae that were not identified in previous analyses of the genome.