Aaron A. Hoskins

Ordered and Dynamic Assembly of Single Spliceosomes

Fluorescently labeled yeast spliceosome proteins reveal the events of intron splicing as it happens. The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

The spliceosome: a flexible, reversible macromolecular machine.

With more than a hundred individual RNA and protein parts and a highly dynamic assembly and disassembly pathway, the spliceosome is arguably the most complicated macromolecular machine in the eukaryotic cell. This complexity has made kinetic and mechanistic analysis of splicing incredibly challenging. Yet, recent technological advances are now providing tools for understanding this process in much greater detail. Ranging from genome-wide analyses of splicing and creation of an orthogonal spliceosome in vivo, to purification of active spliceosomes and observation of single molecules in vitro, such new experimental approaches are yielding significant insight into the inner workings of this remarkable machine. These experiments are rewriting the textbooks, with a new picture emerging of a dynamic, malleable machine heavily influenced by the identity of its pre-mRNA substrate.

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5′ splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.

New insights into the spliceosome by single molecule fluorescence microscopy.

Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ∼100 proteins minimally required for activity. Because of the spliceosomes size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing.

Strategies from UW-Madison for rescuing biomedical research in the US

A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US. DOI: http://dx.doi.org/10.7554/eLife.09305.001

Structural Analysis of Multi-Helical RNAs by NMR–SAXS/WAXS: Application to the U4/U6 di-snRNA

NMR and SAXS (small-angle X-ray scattering)/WAXS (wide-angle X-ray scattering) are highly complementary approaches for the analysis of RNA structure in solution. Here we describe an efficient NMR-SAXS/WAXS approach for structural investigation of multi-helical RNAs. We illustrate this approach by determining the overall fold of a 92-nt 3-helix junction from the U4/U6 di-snRNA. The U4/U6 di-snRNA is conserved in eukaryotes and is part of the U4/U6.U5 tri-snRNP, a large ribonucleoprotein complex that comprises a major subunit of the assembled spliceosome. Helical orientations can be determined by X-ray scattering data alone, but the addition of NMR RDC (residual dipolar coupling) restraints improves the structure models. RDCs were measured in two different external alignment media and also by magnetic susceptibility anisotropy. The resulting alignment tensors are collinear, which is a previously noted problem for nucleic acids. Including WAXS data in the calculations produces models with significantly better fits to the scattering data. In solution, the U4/U6 di-snRNA forms a 3-helix junction with a planar Y-shaped structure and has no detectable tertiary interactions. Single-molecule Förster resonance energy transfer data support the observed topology. A comparison with the recently determined cryo-electron microscopy structure of the U4/U6.U5 tri-snRNP illustrates how proteins scaffold the RNA and dramatically alter the geometry of the U4/U6 3-helix junction.

Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.

SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast

Abstract RNA and protein components of the spliceosome work together to identify the 5΄ splice site, the 3΄ splice site, and the branchsite (BS) of nascent pre-mRNA. SF3b1 plays a key role in recruiting the U2 snRNP to the BS. Mutations in human SF3b1 have been linked to many diseases such as myelodysplasia (MDS) and cancer. We have used SF3b1 mutations associated with MDS to interrogate the role of the yeast ortholog, Hsh155, in BS selection and splicing. These alleles change how the spliceosome recognizes the BS and alter splicing when nonconsensus nucleotides are present at the −2, −1 and +1 positions relative to the branchpoint adenosine. This indicates that changes in BS usage observed in humans with SF3b1 mutations may result from perturbation of a conserved mechanism of BS recognition. Notably, different HSH155 alleles elicit disparate effects on splicing: some increase the fidelity of BS selection while others decrease fidelity. Our data support a model wherein conformational changes in SF3b1 promote U2 association with the BS independently of the action of the DEAD-box ATPase Prp5. We propose that SF3b1 functions to stabilize weak U2/BS duplexes to drive spliceosome assembly and splicing.

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation.

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation. DOI: http://dx.doi.org/10.7554/eLife.14166.001

Formylglycinamide ribonucleotide amidotransferase from Thermotoga maritima: structural insights into complex formation.

In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, Pi, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In Bacillus subtilis, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from Thermotoga maritima is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified.