The septin family of GTPases: architecture and dynamics.

Septins comprise a conserved family of proteins that are found primarily in fungi and animals. These GTP-binding proteins have several roles during cell division, cytoskeletal organization and membrane-remodelling events. One factor that is crucial for their functions is the ordered assembly of individual septins into oligomeric core complexes that, in turn, form higher-order structures such as filaments, rings and gauzes. The molecular details of these interactions and the mechanism by which septin-complex assembly is regulated have remained elusive. Recently, the first detailed structural views of the septin core have emerged, and these, along with studies of septin dynamics in vivo, have provided new insight into septin-complex assembly and septin function in vivo.

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis.

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.

rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly.

Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain (spb1D52A) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF4- transition state structure that implicates the direct involvement of unmodified G2922 in Nog2 GTPase activation. Genetic suppressors and in vivo imaging indicate that premature GTP hydrolysis prevents the efficient binding of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 recruitment to the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Our approach and findings provide a template to study the GTPase cycles and regulatory factor interactions of the other K-loop GTPases involved in ribosome assembly.

The DEAD-box ATPase Dbp10/DDX54 initiates peptidyl transferase center formation during 60S ribosome biogenesis.

DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.

Structure of the C-terminus of the mRNA export factor Dbp5 reveals the interaction surface for the ATPase activator Gle1.

The DExD/H-box RNA-dependent ATPase Dbp5 plays an essential role in the nuclear export of mRNA. Dbp5 localizes to the nuclear pore complex, where its ATPase activity is stimulated by Gle1 and its coactivator inositol hexakisphosphate. Here, we present the crystal structure of the C-terminal domain of Dbp5, refined to 1.8 A. The structure reveals a RecA-like fold that contains two defining characteristics not present in other structurally characterized DExD/H-box proteins: a C-terminal alpha-helix and a loop connecting beta5 and alpha4, both of which are composed of conserved and unique elements in the Dbp5 primary sequence. Using structure-guided mutagenesis, we have identified several charged surface residues that, when mutated, weaken the binding of Gle1 and inhibit the ability of Gle1 to stimulate Dbp5's ATPase activity. In vivo analysis of the same mutations reveals that those mutants displaying the weakest ATPase stimulation in vitro are also unable to support yeast growth. Analysis of the correlation between the in vitro and in vivo data indicates that a threshold level of Dbp5 ATPase activity is required for cellular mRNA export that is not met by the unstimulated enzyme, suggesting a possible mechanism by which Dbp5's activity can be modulated to regulate mRNA export.

Sequence-specific remodeling of a topologically complex RNP substrate by Spb4.

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-specific remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize Spb4 in a post-catalytic, high-energy intermediate that drives the organization of the three-way junction at the base of rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

Eukaryotic Ribosome Assembly and Nuclear Export.

Accurate translation of the genetic code into functional polypeptides is key to cellular growth and proliferation. This essential process is carried out by the ribosome, a ribonucleoprotein complex of remarkable size and intricacy. Although the structure of the mature ribosome has provided insight into the mechanism of translation, our knowledge regarding the assembly, quality control, and intracellular targeting of this molecular machine is still emerging. Assembly of the eukaryotic ribosome begins in the nucleolus and requires more than 350 conserved assembly factors, which transiently associate with the preribosome at specific maturation stages. After accomplishing their tasks, early-acting assembly factors are released, preparing preribosomes for nuclear export. Export competent preribosomal subunits are transported through nuclear pore complexes into the cytoplasm, where they undergo final maturation steps, which are closely connected to quality control, before engaging in translation. In this chapter, we focus on the final events that commit correctly assembled ribosomal subunits for translation.

Impact of rapid response system implementation on critical deterioration events in children.

IMPORTANCE: Rapid response systems aim to identify and rescue deteriorating hospitalized patients. Previous pediatric rapid response system implementation studies have shown variable effectiveness in preventing rare, catastrophic outcomes such as cardiac arrest and death. OBJECTIVE: To evaluate the impact of pediatric rapid response system implementation inclusive of a medical emergency team and an early warning score on critical deterioration, a proximate outcome defined as unplanned transfer to the intensive care unit with noninvasive or invasive mechanical ventilation or vasopressor infusion in the 12 hours after transfer. DESIGN, SETTING, AND PARTICIPANTS: Quasi-experimental study with interrupted time series analysis using piecewise regression. At an urban, tertiary care children's hospital in the United States, we evaluated 1810 unplanned transfers from the general medical and surgical wards to the pediatric and neonatal intensive care units that occurred during 370,504 non-intensive care patient-days between July 1, 2007, and May 31, 2012. INTERVENTIONS: Implementation of a hospital-wide rapid response system inclusive of a medical emergency team and an early warning score in February 2010. MAIN OUTCOMES AND MEASURES: Rate of critical deterioration events, adjusted for season, ward, and case mix. RESULTS: Rapid response system implementation was associated with a significant downward change in the preintervention trajectory of critical deterioration and a 62% net decrease relative to the preintervention trend (adjusted incidence rate ratio = 0.38; 95% CI, 0.20-0.75). We observed absolute reductions in ward cardiac arrests (from 0.03 to 0.01 per 1000 non-intensive care patient-days) and deaths during ward emergencies (from 0.01 to 0.00 per 1000 non-intensive care patient-days), but these were not statistically significant (P = .21 and P = .99, respectively). Among all unplanned transfers, critical deterioration was associated with a 4.97-fold increased risk of death (95% CI, 3.33-7.40; P < .001). CONCLUSIONS AND RELEVANCE: Rapid response system implementation reversed an increasing trend of critical deterioration. Cardiac arrest and death were extremely rare at baseline, and their reductions were not statistically significant despite using nearly 5 years of data. Hospitals seeking to measure rapid response system performance may consider using valid proximate outcomes like critical deterioration in addition to rare, catastrophic outcomes.

Referral patterns and service utilization in a pediatric hospital-wide intimate partner violence program.

OBJECTIVES: To describe the referral patterns and utilization of on-site intimate partner violence (IPV) services in both inpatient and outpatient settings at a large urban children's hospital. METHODS: Retrospective review of case records from IPV victims referred to an on-site IPV counselor between September 2005 and February 2010. Descriptive statistics were used to examine IPV victim demographics, number of referrals per hospital department, referral source (type of staff member), time spent by IPV counselor for initial consultation, and services provided to IPV victims. RESULTS: A total of 453 unique referrals were made to the IPV counselor: 81% were identified by universal screening and 19% by risk-based screening. Thirty-six percent of IPV victims were referred from primary care clinics; 26% from inpatient units; 13% from outpatient subspecialty clinics; 12.5% from the emergency department; 5% from the Child Protection Program; and 4% were employee self-referrals. Social workers generated the most referrals (55%), followed by attending physicians (17%), residents (13%), nurses (7%), and other individuals (self-referrals) (4%). The median initial IPV intervention required 42 minutes. Supportive counseling and safety planning were the services most often utilized by IPV victims. CONCLUSIONS: IPV screening can be successfully integrated in both inpatient and outpatient settings by a multidisciplinary group of hospital staff. Most referrals were generated by universal screening outside of the primary care setting. IPV victims generally desired supportive counseling and safety planning over immediate housing relocation. Many IPV screening opportunities were missed by using verbal screening alone.

Beyond statistical prediction: qualitative evaluation of the mechanisms by which pediatric early warning scores impact patient safety.

BACKGROUND: Early warning scores (EWSs) assign points to clinical observations and generate scores to help clinicians identify deteriorating patients. Despite marginal predictive accuracy in retrospective datasets and a paucity of studies prospectively evaluating their clinical effectiveness, pediatric EWSs are commonly used. OBJECTIVE: To identify mechanisms beyond their statistical ability to predict deterioration by which physicians and nurses use EWSs to support their decision making. DESIGN: Qualitative study. SETTING: A children's hospital with a rapid response system. PARTICIPANTS: Physicians and nurses who recently cared for patients with false-positive and false-negative EWSs (score failures). INTERVENTION: Semistructured interviews. MEASUREMENTS: Themes identified through grounded theory analysis. RESULTS: Four themes emerged among the 57 subjects interviewed: (1) The EWS facilitates safety by alerting physicians and nurses to concerning changes and prompting them to think critically about deterioration. (2) The EWS provides less-experienced nurses with vital sign reference ranges. (3) The EWS serves as evidence that empowers nurses to overcome barriers to escalating care. (4) In stable patients, those with baseline abnormal physiology, and those experiencing neurologic deterioration, the EWS may not be helpful. CONCLUSIONS: Although pediatric EWSs have marginal performance when applied to datasets, clinicians who recently experienced score failures still considered them valuable to identify deterioration and transcend hierarchical barriers. Combining an EWS with a clinician's judgment may result in a system better equipped to respond to deterioration than retrospective data analyses alone would suggest. Future research should seek to evaluate the clinical effectiveness of EWSs in real-world settings.

The Caenorhabditis elegans septin complex is nonpolar.

Septins are conserved GTPases that form heteromultimeric complexes and assemble into filaments that play a critical role in cell division and polarity. Results from budding and fission yeast indicate that septin complexes form around a tetrameric core. However, the molecular structure of the core and its influence on the polarity of septin complexes and filaments is poorly defined. The septin complex of the nematode Caenorhabditis elegans is formed entirely by the core septins UNC-59 and UNC-61. We show that UNC-59 and UNC-61 form a dimer of coiled-coil-mediated heterodimers. By electron microscopy, this heterotetramer appears as a linear arrangement of four densities representing the four septin subunits. Fusion of GFP to the N termini of UNC-59 and UNC-61 and subsequent electron microscopic visualization suggests that the sequence of septin subunits is UNC-59/UNC-61/UNC-61/UNC-59. Visualization of GFP extensions fused to the extremity of the C-terminal coiled coils indicates that these extend laterally from the heterotetrameric core. Together, our study establishes that the septin core complex is symmetric, and suggests that septins form nonpolar filaments.

The N-terminal domain of Nup159 forms a beta-propeller that functions in mRNA export by tethering the helicase Dbp5 to the nuclear pore.

Nuclear export of mRNA in eukaryotic cells is mediated by soluble transport factors and components of the nuclear pore complex (NPC). The cytoplasmically oriented nuclear pore protein Nup159 plays a critical role in mRNA export through its conserved N-terminal domain (NTD). Here, we report the crystal structure of the Nup159 NTD, refined to 2.5 A. The structure reveals an unusually asymmetric seven-bladed beta-propeller that is structurally conserved throughout eukarya. Using structure-based conservation analysis, we have targeted specific surface residues for mutagenesis. Residue substitutions in a conserved loop of the NTD abolish in vitro binding to Dbp5, a DEAD box helicase required for mRNA export. In vivo, these mutations cause Dbp5 mislocalization and block mRNA export. These findings suggest that the Nup159 NTD functions in mRNA export as a binding platform, tethering shuttling Dbp5 molecules at the nuclear periphery and locally concentrating this mRNA remodeling factor at the cytoplasmic face of the NPC.

Localized recruitment of a chromatin-remodeling activity by an activator in vivo drives transcriptional elongation.

To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mouse hsp70 gene. A mutation that diminished transcriptional initiation led to twofold reductions in hsp70 mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from the hsp70 promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-length hsp70 mRNA.