Lights, camera, action! Capturing the spliceosome and pre‐<scp>mRNA</scp> splicing with single‐molecule fluorescence microscopy

DeHaven, Alexander C.; Norden, Ian S.; Hoskins, Aaron A.

doi:10.1002/wrna.1358

Cited by 9 publications

(7 citation statements)

References 124 publications

(294 reference statements)

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“…The supraspliceosome model is consistent with most imaging studies, and different interpretations can account for the limited discrepancies. For example, the differences in K o ff between the U snRNPs were interpreted as supporting the step‐wise assembly model . However, an alternative interpretation of these results is that the binding constants of U1 and U4 snRNPs to the spliceosome are different from those of U2 and U5 snRNPs, and not necessarily reflecting their loss from the spliceosome.…”

Section: The Supraspliceosomementioning

confidence: 92%

“…For example, the differences in Ko ff between the U snRNPs were interpreted as supporting the step-wise assembly model. 38 However, an alternative interpretation of these results is that the binding constants of U1 and U4 snRNPs to the spliceosome are different from those of U2 and U5 snRNPs, and not necessarily reflecting their loss from the spliceosome. Indeed, U1 is less tightly bound to the spliceosome, and is released from complex B upon treatment with heparin.…”

Section: The Supraspliceosome Modelmentioning

confidence: 99%

“…One way to study the in vivo spliceosome is through visualization of splicing and the spliceosome in intact and live cells. An important step in this direction was the development of genetically encoded fluorescent protein tags and high‐resolution microscopes (reviewed in Refs ).…”

Section: The Endogenous Spliceosome –Imaging Studiesmentioning

confidence: 99%

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The nuts and bolts of the endogenous spliceosome

Sperling

2016

WIREs RNA

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The complex life of pre-mRNA from transcription to the production of mRNA that can be exported from the nucleus to the cytoplasm to encode for proteins entails intricate coordination and regulation of a network of processing events. Coordination is required between transcription and splicing and between several processing events including 5' and 3' end processing, splicing, alternative splicing and editing that are major contributors to the diversity of the human proteome, and occur within a huge and dynamic macromolecular machine-the endogenous spliceosome. Detailed mechanistic insight of the splicing reaction was gained from studies of the in vitro spliceosome assembled on a single intron. Because most pre-mRNAs are multiintronic that undergo alternative splicing, the in vivo splicing machine requires additional elements to those of the in vitro machine, to account for all these diverse functions. Information about the endogenous spliceosome is emerging from imaging studies in intact and live cells that support the cotranscriptional commitment to splicing model and provide information about splicing kinetics in vivo. Another source comes from studies of the in vivo assembled spliceosome, isolated from cell nuclei under native conditions-the supraspliceosome-that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Recent years have portrayed new players affecting alternative splicing and novel connections between splicing, transcription and chromatin. The challenge ahead is to elucidate the structure and function of the endogenous spliceosome and decipher the regulation and coordination of its network of processing activities. WIREs RNA 2017, 8:e1377. doi: 10.1002/wrna.1377 For further resources related to this article, please visit the WIREs website.

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Section: The Supraspliceosomementioning

confidence: 92%

Section: The Supraspliceosome Modelmentioning

confidence: 99%

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The nuts and bolts of the endogenous spliceosome

Sperling

2016

WIREs RNA

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“…The active spliceosome assembles through a series of discrete, ATP-dependent conformational transitions that are separable by native gel electrophoresis [108] and also have been illuminated by single molecule fluorescence (reviewed in [109]). The “E”-complex of the U1 snRNP and U2AF–SF1 complex at the 5′ and 3′ splice sites is followed by the ATP-dependent “A”-complex, in which the SF3B-containing U2 snRNP stably associates.…”

Section: Recurrent Somatic Mutations Of Pre-mrna Splicing Factors In mentioning

confidence: 99%

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures

Jenkins

Kielkopf

2017

Trends in Genetics

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Somatic mutations of pre-mRNA splicing factors recur among patients with myelodysplastic syndromes (MDS) and related malignancies. Although these MDS-relevant mutations alter splicing of a subset of transcripts, the mechanisms by which these single amino acid substitutions change gene expression remain controversial. New structures of spliceosome intermediates and associated protein complexes shed light on the molecular interactions mediated by “hotspots” of the SF3B1 and U2AF1 pre-mRNA splicing factors. The frequently mutated SF3B1 residues contact the pre-mRNA splice site. Based on structural-homology with other spliceosome subunits and recent findings of altered RNA binding by mutant U2AF1 proteins, we suggest that affected U2AF1 residues also contact pre-mRNA. Altered pre-mRNA recognition emerges as a molecular theme among MDS-relevant mutations of pre-mRNA splicing factors.

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“…Despite decades of study, many fundamentals of the spliceosome function have remained elusive. Single-molecule detection methods enabled investigators to monitor the dynamics of specific splicing components, whole spliceosomes, and even cotranscriptional splicing within living cells (DeHaven et al, 2016).…”

Section: Applications Of Single-molecule Techniques In Biologymentioning

confidence: 99%

Single-Molecule Science

2022

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Single Molecule Science (SMS) has emerged from developing, using and combining technologies such as super-resolution microscopy, atomic force microscopy, and optical and magnetic tweezers, alongside sophisticated computational and modelling techniques. This comprehensive, edited volume brings together authoritative overviews of these methods from a biological perspective, and highlights how they can be used to observe and track individual molecules and monitor molecular interactions in living cells. Pioneers in this fast-moving field cover topics such as single molecule optical maps, nanomachines, and protein folding and dynamics. A particular emphasis is also given to mapping DNA molecules for diagnostic purposes, and the study of gene expression. With numerous illustrations, this book reveals how SMS has presented us with a new way of understanding life processes. A must-have for researchers and graduate students, as well as those working in industry, primarily in the areas of biophysics, biological imaging, genomics and structural biology.

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Lights, camera, action! Capturing the spliceosome and pre‐mRNA splicing with single‐molecule fluorescence microscopy

Cited by 9 publications

References 124 publications

The nuts and bolts of the endogenous spliceosome

The nuts and bolts of the endogenous spliceosome

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures

Single-Molecule Science

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