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.

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP 6 )-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP 6 stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/ R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42D double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP 6 regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

Chronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV‐mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus‐associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA‐binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD box protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2‐mediated gene repression, acting by regulating RNA‐protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2‐repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized SUZ12‐ and PRC2‐mediated gene silencing, by displacing the RNA‐binding E3 ligase, Mex‐3 RNA‐binding family member B (Mex3b), from HOTAIR. Conversely, ectopic expression of Mex3b ubiquitinated SUZ12, displaced DDX5 from HOTAIR, and induced SUZ12 down‐regulation. In G2 phase of cells expressing the HBV X protein (HBx), SUZ12 preferentially associated with Mex3b, but not DDX5, resulting in de‐repression of PRC2 targets, including EpCAM and pluripotency genes. Significantly, liver tumors from HBx/c‐myc bitransgenic mice and chronically HBV‐infected patients exhibited a strong negative correlation between DDX5 messenger RNA levels, pluripotency gene expression, and liver tumor differentiation. Notably, chronically infected HBV patients with HCC expressing reduced DDX5 exhibited poor prognosis after tumor resection, identifying DDX5 as an important player in poor prognosis HCC. Conclusion: The RNA helicase DDX5, and E3 ligase Mex3b, are important cellular targets for the design of novel, epigenetic therapies to combat HBV infection and poor prognosis HBV‐associated liver cancer. (Hepatology 2016;64:1033‐1048)

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP 6 ). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP 6 /Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.[Keywords: nucleocytoplasmic transport; DEAD-box proteins; nuclear pore complex; FRAP; dominant-negative mutants] Supplemental material is available for this article. Messenger RNAs (mRNAs) are produced in the nucleus through a series of highly coordinated steps that include transcription, processing, and assembly with proteins to form a messenger ribonucleoprotein complex (mRNP) (for reviews, see Pandit et al. 2008;Zhong et al. 2009; Licatalosi and Darnell 2010). Some of the factors that participate in these steps associate with RNA polymerase II during transcription (Cho et al. 1997;Moore and Proudfoot 2009;Perales and Bentley 2009), facilitating coordination by positioning these factors to interact with nascent RNAs upon recognition of key sequence elements or structures. This results in the formation of mRNPs that are exported to the cytoplasm for translation after premRNA processing has been completed. mRNPs are exported through nuclear pore complexes (NPCs), large macromolecular structures (>60 MDa) embedded in the nuclear envelope (NE) (for reviews, see D'Angelo and Hetzer 2008;Hetzer and Wente 2009;Wente and Rout 2010). NPCs have eightfold radial symmetry perpendicular to the NE plane, and the NPC core has twofold symmetry in the NE plane. Attached to the core