Conformational dynamics of single pre-mRNA molecules during in vitro splicing

Abelson, John; Blanco, Mario R.; Ditzler, Mark A.; Fuller, Franklin D.; Aravamudhan, Pavithra; Wood, Mona L.; Villa, Tommaso; Ryan, Daniel E.; Pleiss, Jeffrey A.; Maeder, Corina; Guthrie, Christine; Walter, Nils G.

doi:10.1038/nsmb.1767

Cited by 86 publications

(141 citation statements)

References 38 publications

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“…2D); these excursions might correspond to the close approach detected in trapping studies. Transient fluctuations in single-molecule E FRET were also observed in a previous splicing study (20). However, the relationship between those observations and ours is unclear because the D and A positions were different, and the earlier experiments were conducted with higher time resolution but shorter data record durations.…”

Section: Discussionsupporting

confidence: 42%

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

Crawford

Hoskins

Friedman

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Removal of introns from the precursors to messenger RNA (premRNAs) 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.splicing mechanism | single-molecule FRET | RNA dynamics I ntron excision from precursors to messenger RNAs (premRNAs) is carried out by the spliceosome, arguably the most complex macromolecular machine in the cell (1). One of the most important jobs of the spliceosome is to accurately and efficiently identify the ends of introns and bring them together to promote the chemistry of splicing. This chemistry occurs via two S N 2 transesterification reactions: (i) attack by the branch site (BS) adenosine on the phosphodiester bond at the beginning of the intron (the 5′ splice site; 5′SS) and (ii) attack of the released 5′ exon on the phosphodiester bond at the end of the intron (the 3′SS) (2). The BS adenosine is internal to the intron and usually located in the vicinity of the 3′SS.The spliceosome consists of four major subcomplexes that must assemble de novo on each new intron: the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), the U4/U6.U5 tri-snRNP, and the protein-only nineteen complex (NTC). The snRNPs each contain numerous proteins and one or more small nuclear RNAs (snRNAs). These subcomplexes assemble stepwise, with U1 and U2 recognition of the 5′SS and BS, respectively, preceding trisnRNP and NTC recruitment (3, 4). Throughout the assembly process, numerous large-scale conformational changes occur that involve making and breaking of pre-mRNA:snRNA and snRNA: snRNA base pairing interactions. These structural transitions are necessary for both recognition of the splice sites and creation of the catalytic core in which the splice sites are juxtaposed for chemistry. The two chemical steps occur within the activated spliceosome formed after ejection of the U1 and U4 snRNPs (5).When during spliceosome assembly are the splice sites brought into close proximity? Previous studies in human and yeast extracts using hydroxy radical cleavage or protein-RNA crosslinking led to the hypothesis that the 5′SS and BS regions are closely positioned in early complexes containing only U1 and/or U2 (6-12). However, as both of these irreversible trapping methods can capture transient excursions that are no...

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Section: Discussionsupporting

confidence: 42%

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

Crawford

Hoskins

Friedman

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…HMM also yields transition density contour maps that we find to be highly symmetric with respect to the main diagonal for all ribozymes and Mg 2+ concentrations, indicative of the fact that most transitions occur reversibly between pairs of FRET states (Fig. 5D-F) as expected for an RNA at thermal equilibrium (Abelson et al 2010). In addition, the maps reveal more frequent (color scale in Fig.…”

Section: Afhh-wt Exhibits the Most Sm-fret States And The Greatest Inmentioning

confidence: 97%

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

McDowell¹,

Jun²,

Walter³

2010

RNA

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Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in~10-fold higher than physiologic concentrations of Mg 2+ ions. Extended versions containing native loop-loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg 2+ concentrations, for reasons that are still ill-understood. Here, we use Mg 2+ titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop-loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop-loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop-loop interactions in extended hammerhead ribozymes-and Mg 2+ ions that bind to minimal ribozymes-may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state.

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“…These builtin proofreading mechanisms have to be complex, and require a multistep process because of the dynamic nature of the spliceosome and of its assembly on the splicing substrates (Wahl et al 2009;Abelson et al 2010), and because at least three substrates are involved in two successive chemical steps (Figs. 1, 2).…”

Section: Kinetic Proofreading Activities Mediated By Spliceosomal Atpmentioning

confidence: 99%

Proofreading and spellchecking: A two-tier strategy for pre-mRNA splicing quality control

Egecioglu

Chanfreau²

2011

RNA

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Multi-tier strategies exist in many biochemical processes to ensure a maximal fidelity of the reactions. In this review, we focus on the two-tier quality control strategy that ensures the quality of the products of the pre-mRNA splicing reactions catalyzed by the spliceosome. The first step in the quality control process relies on kinetic proofreading mechanisms that are internal to the spliceosome and that are performed by ATP-dependent RNA helicases. The second quality control step, spellchecking, involves recognition of unspliced pre-mRNAs or aberrantly spliced mRNAs that have escaped the first proofreading mechanisms, and subsequent degradation of these molecules by degradative enzymes in the nucleus or in the cytoplasm. This two-tier quality control strategy highlights a need for high fidelity and a requirement for degradative activities that eliminate defective molecules. The presence of multiple quality control activities during splicing underscores the importance of this process in the expression of genetic information.

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Conformational dynamics of single pre-mRNA molecules during in vitro splicing

Cited by 86 publications

References 38 publications

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

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

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

Proofreading and spellchecking: A two-tier strategy for pre-mRNA splicing quality control

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