2009-03-29

Dinman 2008JBC

(a review of Ribosome, be good for the report)

Despite having been identified first, their greater degree of complexity has resulted in our understanding of  eukaryotic ribosomes lagging behind that of their bacterial and archael counterparts. A much more complicated biogenesis program results in ribosomes that are structurally, biochemically, and functionally more complex. However, recent advances in molecular genetics and structural biology are helping to reveal the intricacies of the eukaryotic ribosome, and to address many longstanding questions regarding its many roles in regulation of gene expression.

For exemple, while bacterial translation initiation only requires a small set of trans-acting factors and is facilitated by the Shine-Dalgarno sequence, this process in eukaryotes requires a multifactorial complex of trans-acting factors that is almost as massive as the riboslme itself.

2009-03-26

36#Spiller et al. 2007J Cell Science

Sm-like (Lsm) proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many RNAs. In eukaryotes, a hetero-heptameric complex of seven Lsm proteins (Lsm2-8) affects the processing of small stable RNAs and pre-mRNAs in the nucleus, whereas a different hetero-heptameric complex of Lsm proteins (Lsm1-7) promotes mRNA decapping and decay in the cytoplasm. These two complexes have six constituent proteins in common, yet localize to separate cellular compartments and perform apparently disparate functions. Little is known about the biogenesis of the Lsm complexes, or how they are recruited to different cellular compartments. We show that, in yeast, the nuclear accumulation of Lsm proteins depends on complex formation and that the Lsm8p subunit plays a crucial role. The nuclear localization of Lsm8p is itself most strongly influenced by Lsm2p and Lsm4p, its presumed neighboursin the Lsm2-8p complex. Furthermore, overexpression and depletion experiments imply that Lsm1p and Lsm8p act competitively with respect to the localization of the two complexes, suggesting a potential mechanism for coregulation of nuclear and cytoplasmic RNA processing. A shift of Lsm proteins from the nucleus to the cytoplasm under stress conditions indicates that this competition is biologically significant.

lsm8-1 (pannone et al, 1998): causes dramatically decreased levels of Lsm8p, is not lethal, resulting only in a weak growth phenotype.

Synowsky et al.2008Protein Science

The yeast Ski complex assists the exosome in the degradation of mRNA. The Ski complex consists of three components; Ski2, Ski3, and Ski8, believed to be present in a 1:1:1 stoichiometry. Measuring the mass of intact isolated endogenously expressed Ski complexes by native mass spectrometry we unambiguously demonstrate that the Ski complex has a hetero-tetrameric stoichiometry consisting of one copy of Ski2 and Ski3 and two copies of Ski8. To validate the stoichiometry of the Ski complex, we performed tandem mass spectrometry. In these experiments one Ski8 subunit was ejected concomitant with the formation of a Ski2/Ski3/Ski8 fragment, confirming the proposed stoichiometry. To probe the topology of the Ski complex we disrupted the complex and mass analyzed the thus formed subcomplexes, detecting Ski8–Ski8, Ski2–Ski3, Ski8–Ski2, and Ski8–Ski8–Ski2. Combining all data we construct an improved structural model of the Ski complex.

The Ski complex is involved in exosome mediated 3′-5′ mRNA degradation.

2009-02-24

Allen et al. 2002Molcular Cellular Proteomics

The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport. Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways. Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins. The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6–10 different karyopherin bs, including importins as well as exportins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95pzKap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding. The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex.

Nucleoporin Affinity-capture Experiments:

Affinity-capture experiments were performed in binding buffer (20 mM Hepes, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc)2, 2 mM dithiothreitol, 0.1% Tween 20). For each experiment, glutathione-Sepharose beads (beads) (Amersham Pharmacia Biotech) and an E. coli extract containing the desired GST-Nup were incubated for 20 min at 4 °C. Beads were collected by sedimentation at 2000 3 g for 30 s and washed 73 by resuspension and sedimentation; two middle washes contained 0.1 mM ATP followed by two others containing 1 M NaCl. Equal aliquots of beads were then incubated with 1 ml of yeast extract (;10 mg). In some experiments, the yeast extracts were supplemented with 30 mg of HIS-Gsp1p-GTP (Q71L) 15 min prior to mixing with the immobilized GST-Nup. After 2 h at 4 °C, beads were washed 63 with binding buffer (.1 millionfold dilution). Bound proteins were eluted with 100 ml of 1 M NaCl and concentrated by trichloroacetic acid/sodium deoxycholate precipitation. Proteins that resistedsalt extraction were subsequently extracted from the beads with SDS.All protein were resolved by SDS-polyacrylamide gel electrophoresis
(7.5% gels) and visualized with Coomassie Blue.

Allen et al. 2002 Mol Cell. Proteomics

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling  receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA. More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (KD
1.5 M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

Karyopherins:

• Importins: Kap95-Kap60, Kap104, Kap121, Kap123, Mtr10, Nmd5, Sxm1, Pdr6, Kap114, Ntf2;
• Exportins: Crm1, Cse1, Kap120, Los1, Mex67-Mtr2;
• Transportins: Msn5.

Nucleoporins:

• FG Nups: Nup1,Nup2,Nup42,Nup49,Nup53,Nup57,Nup59,Nup60,Nup100,Nup116,Nup145,Nup159,Nsp1;
• Non FG Nups: Nup82,Nup84,Nup85,Nup120,Nup133,Nup145,Nup157,Nup170,Nup188,Nup192,Sec13, Seh1, Cdc31, Nic96;
• Poms: Pom34, Pom152, Ndc1.

2009-03-23

StraBer et al. 2000 JCB

It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore. Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins. For this interaction, complex formation between Mex67p and Mtr2p has to occur. MEX67 and MTR2also  genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p. These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore.

Hung et al. 2006MBC

Arx1 and Rei1 are found on late pre-60S ribosomal particles containing the export adaptor Nmd3. Arx1 is related to methionine aminopeptidases (MetAPs), and Rei1 is a C2H2 zinc finger protein whose function in ribosome biogenesis has not been previously characterized. Arx1 and Rei1 localized predominately to the nucleus and cytoplasm, respectively, but could be coimmunoprecipitated, suggesting that they are transiently in the same 60S complex. arx1
mutants showed a modest accumulation of 60S subunits in the nucleus, suggesting that Arx1 enhances 60S export. Deletion of REI1 led to cold sensitivity and redistribution of Arx1 to the cytoplasm, where it remained bound to free 60S subunits. However, deletion of ARX1 or the fusion of enhanced GFP (eGFP) to Rpl25 suppressed the cold sensitivity of an rei1
mutant. The presence of eGFP on Rpl25 or its neighboring protein Rpl35 reduced the binding of Arx1 to 60S subunits, suggesting that Arx1 binds to 60S subunits in the vicinity of the exit tunnel. Mutations in Arx1 that disrupted its binding to 60S also suppressed an rei1
mutant and restored the normal nuclear localization of Arx1. These results indicate that the cold sensitivity of rei1
cells is due to the persistence of Arx1 on 60S subunits in the cytoplasm. Furthermore, these results suggest that Rei1 is needed for release of Arx1 from nascent 60S subunits after export to the cytoplasm but not for the subsequent nuclear import of Arx1.

2009-03-22

YAO et al. Embo J 2007

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nculear pore complex devoid of Phe-Gly domains. In vitro, pe-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extented Mex67-Mtr2 surface. Chemcal crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre=60S subunit export impared by mutating Mtr2 or the 60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNa and ribosome export pathways.

A voir le protocole de protein expression in E coli et in vitro binding and competition assays.

Bradatsh et al. 2007 Mol Cell

Shuttling transport receptors carry cargo through nuclear pore complexes (NPCs) via transient interactions with phe-Gly (FG)-rich nucleoporins. Here, we identify Arx1, a factor associated with a late 60S preribosomal particle in the nucleus, as an unconventional export receptor. Arx1 binds directly to FG nucleoporins and exhibits facilitated translocation through NPCs. Moreover, Arx1 functionally overlaps with the other 60S export receptors, Xpo1 and Mex67-Mtr2, and is genetically linked to nucleoporins. Unexpectedly, Arx1 is structurally unrelated to known shuttling transport receptors but homologous to methionine aminopeptidases (MetAPs), however, without enzymatic activity. Typically, the MetAP fold creates a central cavity that binds the methionine. In contrast, the predicted central cavity of Arx1 is involved in the interaction with FG repeat nucleoporins and 60S subunit export. Thus, an ancient enzyme fold has been adopted by Arx1 to function as a nuclear export receptor.

A voir: condition de rio2-1Rps2eGFP

Hung et al. MBC2008

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

2009-03-15

P-bodies

Cytoplasmic processing bodies, or P-bodies, are RNA-protein granules found in eukaryotic cells. P-bodies contain non-translating mRNAs and proteins involved in mRNA degradation and traslational repression. P-bodies, and the mRNPs within them, have been implicated in mRNA storage, mRNA degradation, and translational repression. The analysis of mRNA turnover often involves the analysis of P-bodies.

Sm-like proteins (spiller 2007 jounal of cell science)

Lsm proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many RNAs. In eukaryotes, a hetero-heptameric complex of seven Lsm proteins (Lsm2-8) affects the processing of small stable RNAs and pre-mRNAs in the nucleus, whereas a different hetero-heptameric complex of Lsm proteins (Lsm1-7)  promotes mRNA decapping and decay in the cytoplasm. These two complexes have six constituent proteins in common., yet localize to separate cellular compartments and perform apparently disparate functions. Little is known about the biogenesis of the Lsm complexes, or how they are recruited to different cellular compartments. We show that, in yeast, the nuclearaccumulation of Lsm proteins depends on complex formation and that the Lsm8p subunit plays a crucial role. The nuclear localization of Lsm8p is itself most strongly influenced by Lsm2p and Lsm4p, its presumed neighbours in the Lsm2-8p complex. Furthermore, overexpression and depletion experiments imply that Lsm1p and Lsm8p act competitvely with respect to the localization of the two complexes, suggestinga potential mechanism for coregulation of nculear and cytoplasmic RNA processing. A shift of Lsm proteins from the nculeus to the cytoplasm under stress conditions indicates that this  competition is biologically significant.