Visualizing the splicing of single pre-mRNA molecules in whole cell extract

Crawford, Daniel J.; Hoskins, Aaron A.; Friedman, Larry J.; Gelles, Jeff; Moore, Melissa J.

doi:10.1261/rna.794808

Cited by 98 publications

(119 citation statements)

References 36 publications

(51 reference statements)

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“…The recently-attained ability to follow pre-mRNA splicing by FRET at the single molecule level represents a technical breakthrough toward this important goal [50].…”

Section: How Atp-dependent Rna Chaperones Assist Foldingmentioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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SummaryThe rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.The explosion in our knowledge about noncoding RNA (ncRNA) -RNA that is not translated into protein -has expanded interest in RNA beyond the traditional RNA community. Diverse ncRNAs include the recently-discovered riboswitches, whose novel gene-regulation function is induced by the binding of small metabolites [1]; most of the so-called "Human Accelerated Regions" (HAR) of the human genome, whose recent evolution may be linked to the emergence of the human brain [2]; and many ncRNAs of unknown function [3]. These discoveries underscore the need to understand the fundamental macromolecular behavior of RNA, its structure and dynamics, and how these RNAs are handled and exploited in biology.Study of catalytic RNAs, which allow detection of correct folding via activity measurements, has established basic thermodynamic and kinetic properties of RNA folding. Large catalytic RNAs such as the group I intron from Tetrahymena thermophila and the RNase P RNA from Bacillus subtilis fold at vastly different rates under different conditions upon addition of Mg 2+ and have been shown to fold via multiple parallel pathways, in which different molecules follow different routes with different rates and intermediates, to a common final folded structure [4,5]. These observations support the common view that RNAs traverse rugged energy landscapes as they fold [6,7]. However, little is known about the true shape of these

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“…The recently-attained ability to follow pre-mRNA splicing by FRET at the single molecule level represents a technical breakthrough toward this important goal [50].…”

Section: How Atp-dependent Rna Chaperones Assist Foldingmentioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

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“…In pioneering work from Hoskins and coworkers (Crawford et al 2008;Hoskins et al 2011), this process of splicing was reconstituted from yeast whole-cell extracts and visualized with single-molecule microscopy in vitro. The kinetic picture that emerged revealed a number of important features.…”

Section: Splicingmentioning

confidence: 99%

“…These same characteristics that have made splicing refractory to other biochemical approaches provide a unique opportunity for single-molecule approaches. In fact, splicing was originally discovered through single-molecule imaging (Berget et al 1977).In pioneering work from Hoskins and coworkers (Crawford et al 2008;Hoskins et al 2011), this process of splicing was reconstituted from yeast whole-cell extracts and visualized with single-molecule microscopy in vitro. The kinetic picture that emerged revealed a number of important features.…”

mentioning

confidence: 99%

What have single-molecule studies taught us about gene expression?

Chen

Larson

2016

Genes Dev.

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The production of a single mRNA is the result of many sequential steps, from docking of transcription factors to polymerase initiation, elongation, splicing, and, finally, termination. Much of our knowledge about the fundamentals of RNA synthesis and processing come from ensemble in vitro biochemical measurements. Single-molecule approaches are very much in this same reductionist tradition but offer exquisite sensitivity in space and time along with the ability to observe heterogeneous behavior and actually manipulate macromolecules. These techniques can also be applied in vivo, allowing one to address questions in living cells that were previously restricted to reconstituted systems. In this review, we examine the unique insights that single-molecule techniques have yielded on the mechanisms of gene expression.Single-molecule experiments are now pervasive in biology. What started out as an experimental approach for characterizing ion channels in the 1970s (Neher and Sakmann 1976) has now become a fixture in hundreds of laboratories addressing fundamental questions in biochemistry, cell biology, genetics, and development. The methodology is nearly as diverse as the problems that are addressed and encompasses imaging, optical tweezers, atomic force microscopy, electrophysiology, and cryo-electron microscopy (cryo-EM), to list a few. The unifying principle behind these approaches is straightforward: the ability to observe the heterogeneous, rare, or fleeting behavior of macromolecules that is normally masked by ensemble techniques. In this review, we focus on the role that single-molecule approaches have played in advancing our understanding of the early steps in gene expression.In general, transcription by RNA polymerase (RNAP) in bacteria or RNA polymerase II (Pol II) in eukaryotes begins when transcription factors (TFs) are recruited to the promoter. This leads to the assembly of the preinitiation complex (PIC) that contains a semicompetent polymerase. The PIC is necessary for unwinding duplex DNA and setting the stage for processive elongation by the polymerase. After conformational changes in the PIC, the polymerase escapes the promoter region and enters into productive elongation. In eukaryotes, the nascent RNA undergoes further modifications such as addition of a 5 ′ cap, synthesis of a poly-A tail, and splicing before the mature mRNA is formed. These early steps of gene expression are uniquely suited to elucidation through singlemolecule methods. For example, single-molecule experimental approaches allow one to visualize the order of assembly for molecular complexes (i.e., the PIC) and observe the variety of pathways that can result in initiation of RNAP. The ability to observe kinetics in unperturbed systems can provide clues to the mechanisms of transcription. RNAPs can also be manipulated by singlemolecule optical trapping to reveal the inner workings of force generation by this enzyme. Furthermore, singlemolecule imaging has also revealed the heterogeneity of gene expression that exists among cells in ...

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“…Yeast extracts were prepared as described previously (Ansari and Schwer 1995;Crawford et al 2008) except that strains transformed with the pAAH0290 were grown in -URA dropout media.…”

Section: Yeast Extract Preparationmentioning

confidence: 99%

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

Rodgers

Paulson

Hoskins

2015

RNA

Self Cite

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Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments.

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Visualizing the splicing of single pre-mRNA molecules in whole cell extract

Cited by 98 publications

References 36 publications

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

What have single-molecule studies taught us about gene expression?

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

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