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

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

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

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

A framework for explaining the role of values in health policy decision-making in Latin America : a critical interpretive synthesis

Abstract: Background: Although values underpin the goals pursued in health systems, including how health systems benefit the population, it is often not clear how values are incorporated into policy decision-making about health systems. The challenge is to encompass social/citizen values, health system goals, and financial realities and to incorporate them into the policy-making process. This is a challenge for all health systems and of particular importance for Latin American (LA) countries. Our objective was to understand how and under what conditions societal values inform decisions about health system financing in LA countries. Methods: A critical interpretive synthesis approach was utilised for this work. We searched 17 databases in December 2016 to identify articles written in English, Spanish or Portuguese that focus on values that inform the policy process for health system financing in LA countries at the macro and meso levels. Two reviewers independently screened records and assessed them for inclusion. One researcher conceptually mapped the included articles, created structured summaries of key findings from each, and selected a purposive sample of articles to thematically synthesise the results across the domains of agenda-setting/prioritisation, policy development and implementation. Results: We identified 5925 references, included 199 papers, and synthesised 68 papers. We identified 116 values and developed a framework to explain how values have been used to inform policy decisions about financing in LA countries. This framework has four categories – (1) goal-related values (i.e. guiding principles of the health system); (2) technical values (those incorporated into the instruments adopted by policy-makers to ensure a sustainable and efficient health system); (3) governance values (those applied in the policy process to ensure a transparent and accountable process of decision-making); and (4) situational values (a broad category of values that represent competing strategies to make decisions in the health systems, their influence varying according to the four factors)..

SUPPORT Tools for evidence-informed health Policymaking (STP) 14: Organising and using policy dialogues to support evidence-informed policymaking

This article is part of a series written for people responsible for making decisions about health policies and programmes and for those who support these decision makers

Communication Research

Contains reports on six research projects.Rand Corporatio

Re-visiting Meltsner: Policy Advice Systems and the Multi-Dimensional Nature of Professional Policy Analysis

10.2139/ssrn.15462511-2