Essential Protein Factors in pre-mRNA splicing : A Structural Study by Nuclear Magnetic Resonance Spectroscopy

This thesis describes two novel three-dimensional structures and the functional characterization of proteins that play important roles in eukaryotic RNA splicing. These results are discussed in Chapters 1 and 2, while biomolecular NMR techniques that were employed for the structure determination are outlined in Chapter 3. Materials and methods are described in Chapter 4. Chapter 1 presents the solution structure of the Tudor domain of the human Survival of Motor Neuron (SMN) protein and its molecular interaction with the spliceosomal Sm proteins. Sm proteins are common components of small nuclear ribonucleoprotein particles (snRNPs), which are assembled by a protein complex that contains SMN. The structure of the SMN Tudor domain exhibits a five stranded ?-barrel, which resembles the fold of Sm proteins. The Tudor domain of SMN binds to arginine and glycine-rich C-terminal regions of Sm proteins, where it specifically recognizes symmetrically di-methylated arginine residues. The E134K mutant Tudor domain, which corresponds to a human mutation associated with Spinal Muscular Atrophy (SMA), is structurally intact but fails to interact with Sm proteins. This provides an explanation for a molecular defect underlying SMA. In Chapter 2, the structural basis for the molecular recognition between the essential splicing factors SF1 and U2 auxiliary factor 2 (U2AF) is provided. This interaction involves the third RNA recognition motif (RRM3) of the large subunit of U2AF (U2AF65) and the N-terminal 25 residues of SF1. The structure of RRM3 exhibits the classical RNP-type fold, but contains an additional C-terminal helix. SF1 is bound by the helical surface of RRM3, opposite of the canonical RNA binding site. The molecular recognition involves insertion of a conserved tryptophan of SF1 into a hydrophobic binding pocket of RRM3. This interaction is complemented by electrostatic contacts that are mediated by acidic residues of RRM3 and basic amino acids of SF1. Surprisingly, the molecular interface is highly similar to that between the large (U2AF65) and small (U2AF35) subunits of U2AF. This RRM-mediated protein interaction provides an example of how conserved structural folds have evolved different molecular functions

Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs

Molecular basis of FIR-mediated c-myc transcriptional control

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.MRC Grant-in-aid U11757455

The after-effects of youth unemployment: More vulnerable persons are less likely to succeed in Youth Guarantee programmes

This article investigates unemployed adolescents’ success in re-employment programmes. It proposes that not being in employment, education, or training indicates a setback in the achievement of important life goals, which affects mental health and success in re-employment programmes. Adolescents who are more affected by the experience of unemployment will be even less likely to succeed. An analysis of longitudinal archival records of 300 adolescents in a Youth Guarantee apprenticeship scheme confirms the expectations. Adolescents who were more vulnerable during unemployment and who had a worse relationship with their parents when starting the apprenticeship were more likely to drop out within the first year. The effect of age was moderated by relationship quality. The results show that taking the prior experience of not being in employment, education and training into account can offer a new understanding for the success of re-employment programmes. Theoretical and practical implications of the findings are discussed

How does job insecurity affect performance and political outcomes? Social identity plays a role

Can job insecurity, performance and political attitudes be connected? The presented study draws from social identity theory to propose that fearing to lose ones job can threaten a person’s identity as an employed person. This identity threat can then lead people to disengage at their work and also shift their political attitudes. A longitudinal survey study among n = 632 British workers was carried to test these assumptions. Results of time stable, cross-lagged structural equation modelling indicate that people who felt more job insecure, also reported less attachment to the general working population and more similarity to the unemployed population at a later time point. At work, this identity threat was related to less persistency. Outside work, it was related to less endorsement of values of group inequality and a shift in self-identified political standing, more to the politically left. The results illustrate that job insecurity is not only relevant for behavior at work and organizational outcomes, but that they can have wider, societally relevant consequences. By including social identity we offer a theoretically well-established explanatory mechanisms to account for this effect. This study broadens current literature in organizational behavior by connecting it to wider outcomes, outside the work context

How does job insecurity affect performance and political outcomes? Social identity plays a role

Can job insecurity, performance and political attitudes be connected? The presented study draws from social identity theory to propose that fearing to lose ones job can threaten a person’s identity as an employed person. This identity threat can then lead people to disengage at their work and also shift their political attitudes. A longitudinal survey study among n = 632 British workers was carried to test these assumptions. Results of time stable, cross-lagged structural equation modelling indicate that people who felt more job insecure, also reported less attachment to the general working population and more similarity to the unemployed population at a later time point. At work, this identity threat was related to less persistency. Outside work, it was related to less endorsement of values of group inequality and a shift in self-identified political standing, more to the politically left. The results illustrate that job insecurity is not only relevant for behavior at work and organizational outcomes, but that they can have wider, societally relevant consequences. By including social identity we offer a theoretically well-established explanatory mechanisms to account for this effect. This study broadens current literature in organizational behavior by connecting it to wider outcomes, outside the work context

Segurança da informação : o usuário como multiplicador

Orientador : Prof. Jaime WojciechowskiMonografia (especialização) - Universidade Federal do Paraná, Setor de Educação, Curso de Especialização em Educação a DistânciaInclui referênciasResumo: O presente trabalho tem por objetivo chamar a atenção dos usuários para a segurança da informação abordando esta questão em um curso de capacitação na modalidade de Educação a Distância com ações simples que podem evitar preocupações futuras mantendo a integridade e proteção de seus dados e de outros a eles confiados. Para isso serão utilizados exemplos de pequenas falhas cometidas diariamente e que acabam expondo e assim deixando vulnerável a segurança da informação. Será disponibilizado ainda, material complementar informativo e educativo para a correta utilização dos equipamentos que armazenam e manipulam informações dando ao usuário suporte necessário para garantir a integridade e segurança da informação, diminuindo a demanda de chamados técnicos para manutenção.Abstract: This paper aims to draw the attention of users to information security by addressing this issue in a training course in Distance Education mode with simple actions that can prevent future concerns while maintaining the integrity and protection of their data and others they trusted. For this will be used examples of small mistakes made daily and we expose and thus leaving vulnerable to information security. Available later, supplementary material informative and educational for the correct use of equipment that store and manipulate information giving the user support needed to ensure the integrity and security of information, reducing the demand for technical calls for maintenance

Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation.

Multisite phosphorylation regulates many transcription factors, including the serum response factor partner Elk-1. Phosphorylation of the transcriptional activation domain (TAD) of Elk-1 by the protein kinase ERK at multiple sites potentiates recruitment of the Mediator transcriptional coactivator complex and transcriptional activation, but the roles of individual phosphorylation events had remained unclear. Using time-resolved nuclear magnetic resonance spectroscopy, we found that ERK2 phosphorylation proceeds at markedly different rates at eight TAD sites in vitro, which we classified as fast, intermediate, and slow. Mutagenesis experiments showed that phosphorylation of fast and intermediate sites promoted Mediator interaction and transcriptional activation, whereas modification of slow sites counteracted both functions, thereby limiting Elk-1 output. Progressive Elk-1 phosphorylation thus ensures a self-limiting response to ERK activation, which occurs independently of antagonizing phosphatase activity

Perceptions of corruption: An empirical study controlling for survey bias

Survey data on corruption are widely used to construct corruption indices, but the underlying data are hardly questioned. How do individual experiences shape perceptions of corruption? Do more work-engaged respondents perceive corruption as a bigger obstacle to business operations than others? What role does answer bias play in corruption surveys? This article brings together several strands of literature to discuss these questions and test them empirically with survey data from Bangladesh and Sri Lanka. We find that individuals who are more work engaged report corruption as a bigger obstacle. So did respondents who were previously exposed to corruption. We control for possible answer bias by implementing a randomized response technique, and find that corruption tends to be under-reported. The effects of work engagement and prior exposure on corruption are more pronounced when the bias indicator is considered, and again become stronger once we control for answer bias affecting past experiences with corruption

In-Cell Protein Structures from 2D NMR Experiments

In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (∼50 μM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures