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. Ecm2 is a non-essential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5' splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing non-consensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the 1st-step catalytic site, promoting 2nd-step catalysis, and permiting alternate 5' SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

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. In this review, we synthesize these and other data with respect to a view of U2 as an assembly of interconnected functional modules. These modules are organized by the U2 small nuclear RNA (snRNA) for roles in spliceosome assembly, intron substrate recognition, and protein scaffolding. We describe new discoveries regarding the structure of U2 components and how the snRNP undergoes numerous conformational and compositional changes during splicing. We specifically highlight large scale movements of U2 modules as the spliceosome creates and rearranges its active site. U2 serves as a compelling example for how cellular machines can exploit the modular organization and structural plasticity of an RNP.

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. Prp5 is essential for stable association of the U2 snRNP with the intron branch site (BS) sequence during spliceosome assembly. Our data show that the Prp5 RecA-like domains undergo a large conformational rearrangement only in response to binding of both ATP and RNA. Mutations in Prp5 impact the fidelity of BS recognition and change the conformational dynamics of the RecA-like domains. We propose that BS recognition during spliceosome assembly involves a set of coordinated conformational switches among U2 snRNP components. Spontaneous toggling of Prp5 into a stable, open conformation may be important for its release from U2 and to prevent competition between Prp5 re-binding and subsequent steps in spliceosome assembly.

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. When smFRET is combined with single-molecule fluorescence colocalization, conformational dynamics can be further linked to the presence or absence of a given spliceosome component. Here, we provide a description of experimental considerations, approaches, and workflows for smFRET with an emphasis on applications for the splicing machinery.

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.

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. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

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. Our analyses reveal that connectivity dynamics result in step-specific impacts of factors on network topology. The spliceosome's connectivity is focused on the active site, in part due to contributions from nonglobular proteins. Many essential factors exhibit large shifts in centralities during splicing. Others show transiently high betweenness centralities at certain stages, thereby suggesting mechanisms for regulating splicing by briefly bridging otherwise poorly connected network nodes. These observations provide insights into organizing principles of the spliceosome and provide frameworks for comparative analysis of other macromolecular machines.

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. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P2(1)2(1)2 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 A. The crystals diffracted X-rays to 2.1 A resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.

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. Stem IIa forms during spliceosome assembly, and stem IIc forms during the catalytic steps. We have studied how uridine 56 and 93 pseudouridylation impacts conformational switching of stem II. Using single-molecule Förster resonance energy transfer, we show that Ψ56 dampens conformational dynamics of stem II and stabilizes stem IIc. In contrast, Ψ93 increases dynamics of non-stem IIc conformations. Pseudouridylation impacts conformational switching of stem II by Mg2+ or the U2 protein Cus2; however, when Mg2+ and Cus2 are used in combination, the impacts of pseudouridylation can be suppressed. These results show that stress-induced post-transcriptional modification of U56 and U93 alters snRNA conformational dynamics by distinct mechanisms and that protein and metal cofactors of the spliceosome alter how snRNAs respond to these modifications.

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. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.