Dynamic and Ordered Assembly of Single Spliceosomes

Hoskins, Aaron A.; Friedman, Larry J.; Gallagher, Sarah S.; Crawford, Daniel J.; Anderson, Eric G.; Wombacher, Richard; Ramirez, Nicholas; Cornish, Virginia W.; Gelles, Jeff; Moore, Melissa J.

doi:10.1016/j.bpj.2010.12.1487

Cited by 79 publications

(139 citation statements)

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“…Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17]. With total internal reflection microscopy ( Figure 1), a localized fluorescence spot on a CCD (charge-coupled device) screen represents the interaction of a fluorescent molecule with a surface-immobilized molecule.…”

Section: Single-molecule Protein Studiesmentioning

confidence: 99%

“…Using cell extracts, Gelles and colleagues visualized spliceosome assembly [14,19], and van Oijen and colleagues reconstituted a eukaryotic replisome on a single-molecule surface [15,20]. Using protein immunoprecipitates, Joo and colleagues have studied the regulation process of microRNA biogenesis [16], and Ha and colleagues have demonstrated single-molecule complex analysis [17,21].…”

Section: Single-molecule Protein Studiesmentioning

confidence: 99%

“…Due to this limitation, the use of cell extracts represents a promising alternative that has been successfully adopted to perform in vitro splicing of labeled pre-mRNA at the singlemolecule level [26,27]. Although it was anticipated that the kinetics of the assembly and action of the spliceosome would be discovered soon after the development and use of single-molecule observations, the dye labeling of spliceosome subunits has only recently been accomplished [14] due to the difficulty of labeling within heterogeneous cell extracts.…”

Section: Real-time Observation Of Spliceosome Assemblymentioning

confidence: 99%

“…However, harsh conditions will induce the dissociation of protein cofactors from protein complexes, making single-molecule IP unsuitable for protein complex studies. Alternatively, problems with antibody specificity can be overcome by preparing immunoprecipitates at higher purities using a tandem purification scheme in which two rounds of immunoprecipitation are carried out using two orthogonal antibodies [16] or by labeling proteins of interest with fluorophores within cell extracts [14,35] …”

Section: Single-molecule Enzymology With Immunoprecipitated Complexesmentioning

confidence: 99%

“…In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al immunostained reaction products using specific fluorescent antibodies [15,20]. …”

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Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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Section: Single-molecule Protein Studiesmentioning

confidence: 99%

Section: Single-molecule Protein Studiesmentioning

confidence: 99%

Section: Real-time Observation Of Spliceosome Assemblymentioning

confidence: 99%

Section: Single-molecule Enzymology With Immunoprecipitated Complexesmentioning

confidence: 99%

mentioning

confidence: 99%

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Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Regulation of alternative mRNA splicing: old players and new perspectives

Dvinge

2018

FEBS Letters

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Nearly all human multiexon genes are subject to alternative splicing in one or more cell types. The splicing machinery therefore has to select between multiple splice sites in a context-dependent manner, relying on sequence features in cis- and trans-acting splicing regulators that either promote or repress splice site recognition and spliceosome assembly. However, the functional coupling between multiple gene regulatory layers signifies that splicing can also be modulated by transcriptional or epigenetic characteristics. Other, less obvious, aspects of alternative splicing have come to light in recent years, often involving core components of the spliceosome previously thought to perform a basal rather than a regulatory role in splicing. Together this paints a highly dynamic picture of splicing regulation, where the final splice site choice is governed by the entire transcriptional environment of a gene and its cellular context.

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Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

Aggarwal

2014

BioEssays

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Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools.

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Dynamic and Ordered Assembly of Single Spliceosomes

Cited by 79 publications

References 0 publications

Bringing single-molecule spectroscopy to macromolecular protein complexes

Bringing single-molecule spectroscopy to macromolecular protein complexes

Regulation of alternative mRNA splicing: old players and new perspectives

Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

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