ACT1-CUP1 Assays Determine the Substrate-specific Sensitivities of Spliceosomal Mutants in Budding Yeast.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level.

Network theory reveals principles of spliceosome structure and dynamics.

Cryoelectron microscopy has revolutionized spliceosome structural biology, and structures representing much of the splicing process have been determined. Comparison of these structures is challenging due to extreme dynamics of the splicing machinery and the thousands of changing interactions during splicing. We have used network theory to analyze splicing factor interactions by constructing structure-based networks from protein-protein, protein-RNA, and RNA-RNA interactions found in eight different spliceosome structures.

Saccharomyces cerevisiae Ecm2 Modulates the Catalytic Steps of pre-mRNA Splicing.

Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how non-essential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery.

Structural and functional modularity of the U2 snRNP in pre-mRNA splicing.

The U2 small nuclear ribonucleoprotein (snRNP) is an essential component of the spliceosome, the cellular machine responsible for removing introns from precursor mRNAs (pre-mRNAs) in all eukaryotes. U2 is an extraordinarily dynamic splicing factor and the most frequently mutated in cancers. Cryo-electron microscopy (cryo-EM) has transformed our structural and functional understanding of the role of U2 in splicing.

Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly.

The DEAD-box family of proteins are ATP-dependent, RNA-binding proteins implicated in many aspects of RNA metabolism. Pre-mRNA splicing in eukaryotes requires three DEAD-box ATPases (Prp5, Prp28 and Sub2), the molecular mechanisms of which are poorly understood. Here, we use single molecule FRET (smFRET) to study the conformational dynamics of yeast Prp5.

Stress-induced Pseudouridylation Alters the Structural Equilibrium of Yeast U2 snRNA Stem II.

In yeast, the U2 small nuclear ribonucleic acid (snRNA) component of the spliceosome is targeted for additional post-transcriptional modifications in response to cellular stress. Uridines 56 and 93 are both modified to pseudouridines (Ψ) during nutrient deprivation, while U56 is also pseudouridylated during heat shock. Both positions are located within stem II, which must toggle between two mutually exclusive structures during splicing.

Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET.

The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub-populations of molecules with unique behaviors.

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics.

Architecture of the spliceosome.

Precursor-mRNA splicing is catalyzed by an extraordinarily large and highly dynamic macromolecular assemblage termed the spliceosome. Detailed biochemical and structural study of the spliceosome presents a formidable challenge, but there has recently been significant progress made on this front highlighted by the crystal structure of a 10-subunit human U1 snRNP. This review provides an overview of our current understanding of the architecture of the spliceosome and the RNA-protein complexes integral to its function, the U snRNPs.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygen.

Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (>2 kU mg(-1)) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction.