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Roy Parker

Publications and source records attributed to Roy Parker.

At least 37 records · Page 2Linked to original sources
PubMed2005

MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies.

Small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs) can silence target genes through several different effector mechanisms. Whereas siRNA-directed mRNA cleavage is increasingly understood, the mechanisms by which miRNAs repress protein synthesis are obscure. Recent studies have revealed the existence of specific cytoplasmic foci, referred to herein as processing bodies (P-bodies), which contain untranslated mRNAs and can serve as sites of mRNA degradation. Here we demonstrate that Argonaute proteins--the signature components of the RNA interference (RNAi) effector complex, RISC--localize to mammalian P-bodies. Moreover, reporter mRNAs that are targeted for translational repression by endogenous or exogenous miRNAs become concentrated in P-bodies in a miRNA-dependent manner. These results provide a link between miRNA function and mammalian P-bodies and suggest that translation repression by RISC delivers mRNAs to P-bodies, either as a cause or as a consequence of inhibiting protein synthesis.

3' Untranslated Regions↗
PubMed2005

Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping.

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.

Apoproteins↗
PubMed2005

Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection.

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p-7p complex is a general activator of decapping and also functions in protecting the 3' ends of deadenylated mRNAs from a 3'-trimming reaction. Lsm1p is the unique member of the Lsm1p-7p complex, distinguishing that complex from the functionally different Lsm2p-8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p-7p complex and the ability of the Lsm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p-7p complex to the P-bodies; (iii) mRNA 3'-end protection could be indicative of the binding of the Lsm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3' end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function.

Base Sequence↗
PubMed2005

The yeast EDC1 mRNA undergoes deadenylation-independent decapping stimulated by Not2p, Not4p, and Not5p.

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.

3' Untranslated Regions↗
PubMed2005

Processing bodies require RNA for assembly and contain nontranslating mRNAs.

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. However, the physical nature of P-bodies, their relationship to translation, and possible roles of P-bodies in cellular responses remain unclear. We describe four properties of yeast P-bodies that indicate that P-bodies are dynamic structures that contain nontranslating mRNAs and function during cellular responses to stress. First, in vivo and in vitro analysis indicates that P-bodies are dependent on RNA for their formation. Second, the number and size of P-bodies vary in response to glucose deprivation, osmotic stress, exposure to ultraviolet light, and the stage of cell growth. Third, P-bodies vary with the status of the cellular translation machinery. Inhibition of translation initiation by mutations, or cellular stress, results in increased P-bodies. In contrast, inhibition of translation elongation, thereby trapping the mRNA in polysomes, leads to dissociation of P-bodies. Fourth, multiple translation factors and ribosomal proteins are lacking from P-bodies. These results suggest additional biological roles of P-bodies in addition to being sites of mRNA degradation.

Cytoplasmic Granules↗
PubMed2004

Crystal structure of Ski8p, a WD-repeat protein with dual roles in mRNA metabolism and meiotic recombination.

Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution. The structure reveals that Ski8p folds into a seven-bladed beta propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed.

Amino Acid Sequence↗
PubMed2004

The yeast Apq12 protein affects nucleocytoplasmic mRNA transport.

An important step in mRNA biogenesis is the export of mRNA from the nucleus to the cytoplasm. In this work, we provide evidence that the previously uncharacterized gene APQ12 functions in nucleocytoplasmic mRNA transport in Saccharomyces cerevisiae. First, apq12delta strains manifest 3' hyperadenylated mRNA similar to other previously characterized RNA export mutants. Second, bulk poly(A)+ RNA is retained in the nucleus in apq12delta cells. Third, an Apq12p-GFP chimeric protein is localized to the nuclear periphery. Fourth, mRNA in apq12delta cells is stabilized, consistent with a defect in the rate of nuclear export. Interestingly, apq12delta mutants are severely compromised for growth and display atypical cell morphology. Because this aberrant cell morphology is not seen with other viable export mutants, Apq12p must have either an additional cellular function, or preferentially impinge on the export of mRNAs regulating cell growth. Together, these findings support a role for APQ12 in nucleocytoplasmic transport of mRNA.

Active Transport, Cell Nucleus↗
PubMed2004

Crystal structure of Dcp1p and its functional implications in mRNA decapping.

A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5'-->3' exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains. Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites. One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins. Moreover, a conserved hydrophobic patch is shown to be critical for decapping.

Amino Acid Sequence↗
PubMed2004

Nonsense-mediated mRNA decay: terminating erroneous gene expression.

Nonsense-mediated mRNA decay is a surveillance pathway that reduces errors in gene expression by eliminating aberrant mRNAs that encode incomplete polypeptides. Recent experiments suggest a working model whereby premature and normal translation termination events are distinct as a consequence of the spatial relationship between the termination codon and mRNA binding proteins, a relationship partially established by nuclear pre-mRNA processing. Aberrant termination then leads to both translational repression and an increased susceptibility of the mRNA to multiple ribonucleases.

3' Untranslated Regions↗
PubMed2004

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. We highlight that there are a variety of enzymes with different specificities, suggesting that individual nucleases act on distinct subpopulations of transcripts within the cell. In several cases, translation factors that bind mRNA inhibit these nucleases. In addition, recent work has begun to identify distinct mRNP complexes that recruit the nucleases to transcripts through different mRNA-interacting proteins. These properties and complexes suggest multiple mechanisms by which mRNA degradation could be regulated.

Adenosine Monophosphate↗
PubMed2004

Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae.

The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5'-3' exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5'-3' degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5'-3' exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Delta had no effect when combined with the lsm1Delta, dhh1Delta, or pat1Delta mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.

Amino Acid Sequence↗
PubMed2004

Eukaryotic mRNA decapping.

Eukaryotic mRNAs are primarily degraded by removal of the 3' poly(A) tail, followed either by cleavage of the 5' cap structure (decapping) and 5'->3' exonucleolytic digestion, or by 3' to 5' degradation. mRNA decapping represents a critical step in turnover because this permits the degradation of the mRNA and is a site of numerous control inputs. Recent analyses suggest decapping of an mRNA consists of four central and related events. These include removal, or inactivation, of the poly(A) tail as an inhibitor of decapping, exit from active translation, assembly of a decapping complex on the mRNA, and sequestration of the mRNA into discrete cytoplasmic foci where decapping can occur. Each of these steps is a demonstrated, or potential, site for the regulation of mRNA decay. We discuss the decapping process in the light of these central properties, which also suggest fundamental aspects of cytoplasmic mRNA physiology that connect decapping, translation, and storage of mRNA.

Codon, Nonsense↗
PubMed2003

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. We demonstrate that the nonsense-containing transcripts are efficiently recognized, targeted for deadenylation-independent decapping, and show NMD triggered accelerated deadenylation regardless of the position of the nonsense codon. We also show that 5' nonsense codons trigger faster rates of decapping than 3' nonsense codons, thereby providing a mechanistic basis for the polar effect of NMD. Finally, we construct a computational model that accurately describes the process of NMD and serves as an explanatory and predictive tool.

Adenine↗
PubMed2003

Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.

A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.

Capsid Proteins↗
PubMed2003

Cytoplasmic degradation of splice-defective pre-mRNAs and intermediates.

Specific systems of nuclear RNA degradation appear to target and degrade aberrant pre-mRNA molecules. In this work we report on a Dbr1p-dependent RNA decay pathway that limits the accumulation of splice-defective lariat intermediates stalled at the second step of splicing. In this pathway, splice-defective lariat intermediates are debranched by Dbr1p and subsequently degraded 5' to 3' primarily by the cytoplasmic exonuclease, Xrn1p. When debranching is blocked, these splicing intermediates can be degraded in a 3' to 5' direction in a manner dependent on Ski2p, a cofactor for the cytoplasmic exosome. In that Xrn1p and Ski2p are cytoplasmic and Dbr1p localizes to both the nucleus and the cytoplasm, these data suggest that this decay pathway occurs within the cytoplasm. Furthermore, the finding that lariat intermediates accumulate in the dbr1Delta strain suggests that this pathway also functions as an inherent quality control mechanism for the process of pre-mRNA splicing.

Cytoplasm↗
PubMed2003

Analysis of recombinant yeast decapping enzyme.

A critical step in the turnover of yeast mRNAs is decapping. Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in the decapping reaction have not been established. To determine the function of both Dcp1p and Dcp2p in decapping, we purified recombinant versions of these proteins from Escherichia coli and examined their properties. These experiments demonstrate that copurification of Dcp1p and Dcp2p yields active decapping enzyme under a variety of conditions. Moreover, Dcp2p alone can have decapping activity under some biochemical conditions. This suggests that Dcp2p can be a catalytic subunit of the decapping complex, and Dcp1p may function to enhance Dcp2p activity, or as an additional active subunit. In addition, recombinant Dcp1p/Dcp2p prefers long mRNA substrates and is sensitive to inhibition by sequestration of the 5' end but not the 3' end of the substrate. This suggests that Dcp1p/Dcp2p contains an additional RNA-binding site spatially distinct from the active site. Finally, using two RNA-binding proteins that enhance decapping in vivo (Edc1p and Edc2p), we can reconstitute the activation of decapping with recombinant proteins. This indicates that the Edc1 and Edc2 proteins act directly on the decapping enzyme.

Endoribonucleases↗
PubMed2003

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. In this work, we demonstrate that Edc1p and Edc2p are RNA-binding proteins. In addition, recombinant Edc1p or Edc2p stimulates mRNA decapping in cell-free extracts or with purified decapping enzyme. These results suggest that Edc1p and Edc2p activate decapping directly by binding to the mRNA substrate and enhancing the activity of the decapping enzyme. Interestingly, edc1Delta strains show defects in utilization of glycerol as a carbon source and misregulation of several mRNAs in response to carbon-source changes. This identifies a critical role for decapping and Edc1p in alterations of gene expression in response to carbon-source changes.

Endoribonucleases↗