PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex

The 2'-5' RNA Ligase of Escherichia coli: Purification, Cloning, and Genomic Disruption

An RNA ligase previously detected in extracts of Escherichia coli is capable of joining Saccharomyces cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2-5 phosphodiester linkage (Greer, C., Javor, B., and Abelson, J. (1983) Cell 33, 899-906). This enzyme specifically ligates tRNA half-molecules containing nucleoside base modifications and shows a preference among different tRNA species. In order to investigate the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from E. coli lysate utilizing chromatographic techniques and separation of proteins by SDS-polyacrylamide gel electrophoresis. A single polypeptide of approximately 20 kilodaltons exhibited RNA ligase activity. The amino terminus of this protein was sequenced, and the open reading frame (ORF) encoding it was identified by a data base search. This ORF, which encodes a novel protein with a predicted molecular mass of 19.9 kDa, was amplified from E. coli genomic DNA and cloned. ORFs coding for highly similar proteins were detected in Methanococcus jannaschii and Bacillus stearothermophilus. The chromosomal gene encoding RNA ligase in E. coli was disrupted, abolishing ligase activity in cell lysates. Cells lacking ligase activity grew normally under laboratory conditions. However, moderate overexpression of the ligase protein led to slower growth rates and a temperature-sensitive phenotype in both wild-type and RNA ligase knockout strains. The RNA ligase reaction was studied in vitro using purified enzyme and was found to be reversible, indicating that this enzyme may perform cleavage or ligation in vivo

In vitro trans-splicing in Saccharomyces cerevisiae

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg^2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency

Impaired contextual modulation of memories in PTSD: an fMRI and psychophysiological study of extinction retention and fear renewal

Post-traumatic stress disorder (PTSD) patients display pervasive fear memories, expressed indiscriminately. Proposed mechanisms include enhanced fear learning and impaired extinction or extinction recall. Documented extinction recall deficits and failure to use safety signals could result from general failure to use contextual information, a hippocampus-dependent process. This can be probed by adding a renewal phase to standard conditioning and extinction paradigms. Human subjects with PTSD and combat controls were conditioned (skin conductance response), extinguished, and tested for extinction retention and renewal in a scanner (fMRI). Fear conditioning (light paired with shock) occurred in one context, followed by extinction in another, to create danger and safety contexts. The next day, the extinguished conditioned stimulus (CS+E) was re-presented to assess extinction recall (safety context) and fear renewal (danger context). PTSD patients showed impaired extinction recall, with increased skin conductance and heightened amygdala activity to the extinguished CS+ in the safety context. However, they also showed impaired fear renewal; in the danger context, they had less skin conductance response to CS+E and lower activity in amygdala and ventral-medial prefrontal cortex compared with combat controls. Control subjects displayed appropriate contextual modulation of memory recall, with extinction (safety) memory prevailing in the safety context, and fear memory prevailing in the danger context. PTSD patients could not use safety context to sustain suppression of extinguished fear memory, but they also less effectively used danger context to enhance fear. They did not display globally enhanced fear expression, but rather showed a globally diminished capacity to use contextual information to modulate fear expression

An engineered Tetrahymena tRNA(Gln) for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology

Protocol for a randomized controlled trial examining multilevel prediction of response to behavioral activation and exposure-based therapy for generalized anxiety disorder.

BACKGROUND:Only 40-60% of patients with generalized anxiety disorder experience long-lasting improvement with gold standard psychosocial interventions. Identifying neurobehavioral factors that predict treatment success might provide specific targets for more individualized interventions, fostering more optimal outcomes and bringing us closer to the goal of "personalized medicine." Research suggests that reward and threat processing (approach/avoidance behavior) and cognitive control may be important for understanding anxiety and comorbid depressive disorders and may have relevance to treatment outcomes. This study was designed to determine whether approach-avoidance behaviors and associated neural responses moderate treatment response to exposure-based versus behavioral activation therapy for generalized anxiety disorder. METHODS/DESIGN:We are conducting a randomized controlled trial involving two 10-week group-based interventions: exposure-based therapy or behavioral activation therapy. These interventions focus on specific and unique aspects of threat and reward processing, respectively. Prior to and after treatment, participants are interviewed and undergo behavioral, biomarker, and neuroimaging assessments, with a focus on approach and avoidance processing and decision-making. Primary analyses will use mixed models to examine whether hypothesized approach, avoidance, and conflict arbitration behaviors and associated neural responses at baseline moderate symptom change with treatment, as assessed using the Generalized Anxiety Disorder-7 item scale. Exploratory analyses will examine additional potential treatment moderators and use data reduction and machine learning methods. DISCUSSION:This protocol provides a framework for how studies may be designed to move the field toward neuroscience-informed and personalized psychosocial treatments. The results of this trial will have implications for approach-avoidance processing in generalized anxiety disorder, relationships between levels of analysis (i.e., behavioral, neural), and predictors of behavioral therapy outcome. TRIAL REGISTRATION:The study was retrospectively registered within 21 days of first participant enrollment in accordance with FDAAA 801 with ClinicalTrials.gov, NCT02807480. Registered on June 21, 2016, before results

Prp43: An RNA helicase-like factor involved in spliceosome disassembly

The Saccharomyces cerevisiae genes PRP2, PRP16, and PRP22 encode pre-mRNA splicing factors that belong to the highly conserved “DEAH” family of putative RNA helicases. We previously identified two additional members of this family, JA1 and JA2. To investigate its biological function, we cloned the JA1 gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family. A ja1 allele carrying a mutation identical to that in the temperature-sensitive (ts) prp22–1 gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement. Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats. Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes. The results presented indicate that JA1 encodes a pre-mRNA processing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA. We have therefore renamed this gene PRP43

Understanding the Role of Values in Health Policy Decision-Making From the Perspective of Policy-Makers and Stakeholders: A Multiple-Case Embedded Study in Chile and Colombia

Background: Chile and Colombia are examples of Latin American countries with health systems shaped by similar values. Recently, both countries have crafted policies to regulate the participation of private for-profit insurance companies in their health systems, but through very different mechanisms. This study asks: what values are important in the decision-making processes that crafted these policies? And how and why are they used?Methods: An embedded multiple-case study design was carried out for 2 specific decisions in each country: (1) in Chile, the development of the Universal Plan of Explicit Entitlements - AUGE/GES - and mandating universal coverage of treatments for high-cost diseases; and (2) in Colombia, the declaration of health as a fundamental right and a mechanism to explicitly exclude technologies that cannot be publicly funded. We interviewed key informants involved in one or more of the decisions and/or in the policy analysis and development process that contributed to the eventual decision. The data analysis involved a constant comparative approach and thematic analysis for each case study. Results: From the 40 individuals who were invited, 28 key informants participated. A tension between 2 important values was identified for each decision (eg, solidarity vs. individualism for the AUGE/GES plan in Chile; human dignity vs. sustainability for the declaration of the right to health in Colombia). Policy-makers used values in the decision-making process to frame problems in meaningful ways, to guide policy development, as a pragmatic instrument to make decisions, and as a way to legitimize decisions. In Chile, values such as individualism and free choice were incorporated in decision-making because attaining private health insurance was seen as an indicator of improved personal economic status. In Colombia, human dignity was incorporated as the core value because the Constitutional Court asserted its importance in its use of judicial activism as a check on the power of the executive and legislative branches.Conclusion: There is an opportunity to open further exploration of the role of values in different health decisions, political sectors besides health, and even other jurisdictions

Channelized melt flow in downwelling mantle: Implications for 226Ra-210Pb disequilibria in arc magmas

We present the results of an analytical model of porous flow of viscous melt into a steadily dilating ‘‘channel’’ (defined as a cluster of smaller veins) in downwelling subarc mantle. The model predicts the pressure drop in the mantle wedge matrix surrounding the channel needed to drive melt flow as a function of position and time. Melt is sucked toward the dilatant region at a near-constant velocity (105 s1) until veins comprising the channel stop opening (t = t). Fluid elements that complete their journey within the time span t < t arrive at a channel. Our results make it possible to calculate the region of influence sampled by melt that surrounds the channel. This region is large compared to the model size of the channelized region driving flow. For a baseline dilation time of 1 year and channel half width of 2 m, melt can be sampled over an 80-m radius and has the opportunity to sample matrix material with potentially contrasting chemistry on geologically short timescales. Our mechanical results are consistent with a downgoing arc mantle wedge source region where melting and melt extraction by porous flow to a channel network are sufficiently rapid to preserve source-derived 238U-230Th-226Ra, and potentially also 226 Ra-210Pb, disequilibria, prior to magma ascent to the surface. Since this is the rate-determining step in the overall process, it allows the possibility that such short-lived disequilibria measured in arc rocks at the surface are derived from deep in the mantle wedge. Stresses due to partial melting do not appear capable of producing the desired sucking effect, while the order of magnitude rate of shear required to drive dilation of 107 s1 is much larger than values resulting from steady state subduction. We conclude that local deformation rates in excess of background plate tectonic rates are needed to ‘‘switch on’’ the dilatant channel network and to initiate the sucking effect

Psychological and Environmental Correlates of HPA Axis Functioning in Parentally Bereaved Children: Preliminary Findings

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97514/1/jts21788.pd