RESUMEN
The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5' splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome.
Asunto(s)
Empalme Alternativo/fisiología , ARN Helicasas DEAD-box/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/fisiología , ARN Helicasas DEAD-box/genética , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/fisiología , ARN Nuclear Pequeño/fisiología , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Empalmosomas/efectos de los fármacos , Empalmosomas/fisiologíaRESUMEN
A primary goal of science and engineering (S&E) education is to produce good problem solvers, but how to best teach and measure the quality of problem solving remains unclear. The process is complex, multifaceted, and not fully characterized. Here, we present a detailed characterization of the S&E problem-solving process as a set of specific interlinked decisions. This framework of decisions is empirically grounded and describes the entire process. To develop this, we interviewed 52 successful scientists and engineers ("experts") spanning different disciplines, including biology and medicine. They described how they solved a typical but important problem in their work, and we analyzed the interviews in terms of decisions made. Surprisingly, we found that across all experts and fields, the solution process was framed around making a set of just 29 specific decisions. We also found that the process of making those discipline-general decisions (selecting between alternative actions) relied heavily on domain-specific predictive models that embodied the relevant disciplinary knowledge. This set of decisions provides a guide for the detailed measurement and teaching of S&E problem solving. This decision framework also provides a more specific, complete, and empirically based description of the "practices" of science.
Asunto(s)
Solución de Problemas , Estudiantes , Ingeniería , HumanosRESUMEN
Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the alpha-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.