The exchange of molecules between the nucleus and cytoplasm is mediated through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Altering the interactions between transport receptors and their cargo has been shown to be a major regulatory mechanism to control traffic through NPCs. New evidence now suggests that NPC proteins play active roles in translocation, and that transport is also controlled by dynamic changes in NPC composition and architecture. This view of ever-changing NPCs necessitates the re-evaluation of current models of nuclear transport and how this process is regulated.

Messenger RNA (mRNA) export involves the unidirectional passage of ribonucleoprotein particles (RNPs) through nuclear pore complexes (NPCs), presumably driven by the ATP-dependent activity of the DEAD-box protein Dbp5. Here we report that Dbp5 functions as an RNP remodeling protein to displace the RNA-binding protein Nab2 from RNA. Strikingly, the ADP-bound form of Dbp5 and not ATP hydrolysis is required for RNP remodeling. In vivo studies with nab2 and dbp5 mutants show that a Nab2-bound mRNP is a physiological Dbp5 target. We propose that Dbp5 functions as a nucleotide-dependent switch to control mRNA export efficiency and release the mRNP from the NPC.

Regulation of nuclear mRNA export is critical for proper eukaryotic gene expression. A key step in this process is the directional translocation of mRNA-ribonucleoprotein particles (mRNPs) through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. Our previous studies in Saccharomyces cerevisiae defined an in vivo role for inositol hexakisphosphate (InsP6) and NPC-associated Gle1 in mRNA export. Here, we show that Gle1 and InsP6 act together to stimulate the RNA-dependent ATPase activity of the essential DEAD-box protein Dbp5. Overexpression of DBP5 specifically suppressed mRNA export and growth defects of an ipk1 nup42 mutant defective in InsP6 production and Gle1 localization. In vitro kinetic analysis showed that InsP6 significantly increased Dbp5 ATPase activity in a Gle1-dependent manner and lowered the effective RNA concentration for half-maximal ATPase activity. Gle1 alone had minimal effects. Maximal InsP6 binding required both Dbp5 and Gle1. It has been suggested that Dbp5 requires unidentified cofactors. We now propose that Dbp5 activation at NPCs requires Gle1 and InsP6. This would facilitate spatial control of the remodelling of mRNP protein composition during directional transport and provide energy to power transport cycles.

Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-speci®c 2¢-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and ®brillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C¢/D¢ RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C¢/D¢ motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, ef®cient ribonucleotide 2¢-O-methylation requires that both the box C/D and C¢/D¢ RNPs function within the fulllength sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C¢/D¢ motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modi®cation complex has resulted in structurally distinct archaeal and eukaryotic RNP particles. Keywords: Archaea/box C/D RNP/ribonucleotide methylation/snoRNA/sRNA IntroductionThe small nucleolar RNAs (snoRNAs) play critical roles in ribosome biogenesis, functioning in the processing and modi®cation of preribosomal RNA (Bachellerie et al., 2002;Kiss, 2002;Terns and Terns, 2002). The primary role of the vast majority of snoRNAs is to guide the sitespeci®c modi®cation of rRNA nucleotides. Guide regions within the snoRNA base pair with complementary sequences in the rRNA and direct snoRNA-associated enzymes to the designate nucleotide for ribose or base modi®cation. Recent work has also revealed guide RNAs in Archaea (Gaspin et al., 2000;Omer et al., 2000). While archaeal organisms do not possess a nucleus, they nevertheless utilize snoRNA-like RNAs (sRNAs) for nucleotide modi®cation. The occurrence of guide RNAs in both Eukarya and Archaea indicates that the process of RNA-guided nucleotide modi®cation is an ancient mechanism predating the divergence of Eukarya and Archaea more than 2 billion years ago.Box C/D RNAs direct the site-speci®c 2¢-O-ribose methylation of targeted nucleotides within rRNA and other RNA substrates (Tollervey, 1996;Tycowski et al., 1998;Jady and Kiss, 2001). Members of this family are de®ned by the conserved boxes C and D located at the 5¢ and 3¢ termini, respectively (Tyc and Steitz, 1989;Caffarelli et al., 1996;Cavaille and Bachellerie, 1996;Watkins et al., 1996Watkins et al., , 2000. These conserved sequences fold into a stem±loop±stem structure which is essential for the binding of box C/D ribonucleoproteins (RNPs) as well as the nucleotide modi®cation reaction itself. Additional internal sequences designated C¢ and D¢ boxes can be identi®ed in eukaryotic snoRNAs and archaeal sRNAs (Kiss-Laszlo et al., 1998). Although based upon boxes C and D, the C¢ and D¢ sequences are not as strictly conserved and are not easily identi®ed in all eukaryotic s...

Summary Gene expression requires proper messenger (m) RNA export and translation. However, the functional links between these consecutive steps have not been fully defined. Gle1 is an essential, conserved mRNA export factor whose export function is dependent on the small molecule inositol hexakisphosphate (IP6). Here we show that both Gle1 and IP6 are required for efficient translation termination in Saccharomyces cerevisiae, and Gle1 interacts with termination factors. In addition, Gle1 has a conserved physical association with the initiation factor eIF3, and gle1 mutants display genetic interactions with the eIF3 mutant nip1-1. Strikingly, gle1 mutants have defects in initiation, whereas strains lacking IP6 do not. We propose that Gle1 functions together with IP6 and the DEAD-box protein Dbp5 to regulate termination. However, Gle1 also independently mediates initiation. Thus, Gle1 is uniquely positioned to coordinate the mRNA export and translation mechanisms. These results directly impact models for perturbation of Gle1 function in pathophysiology.