In vitro assembly of yeast U6 snRNP: a functional assay.

Fabrizio, Patrizia; McPheeters, David S.; Abelson, John

doi:10.1101/gad.3.12b.2137

Cited by 130 publications

(115 citation statements)

References 54 publications

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“…3A) (15). Spliceosome assembly can readily be blocked before U2 addition (16), before U4/U6.U5 addition (17), or before U4 release (18) (Fig. S8).…”

Section: Resultsmentioning

confidence: 99%

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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“…3A) (15). Spliceosome assembly can readily be blocked before U2 addition (16), before U4/U6.U5 addition (17), or before U4 release (18) (Fig. S8).…”

Section: Resultsmentioning

confidence: 99%

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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“…1). The modified U6 RNAs were tested for splicing activity in U6-reconstituted extract using our standard in vitro assay (Fabrizio et al 1989). In addition to the known effect of U6.thioU54 in completely blocking the second chemical step of splicing (Kim and Abelson 1996), another modified U6 RNA, U6.thioU61, was found to inhibit splicing relative to synthetic wild-type U6 RNA control (data not shown).…”

Section: Resultsmentioning

confidence: 99%

New tertiary constraints between the RNA components of active yeast spliceosomes: A photo-crosslinking study

Ryan

Kim²,

Murray³

et al. 2004

RNA

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Elucidation of the three-dimensional (3D) structures of the two sequential active sites in spliceosomes is essential for understanding the mechanism of premessenger RNA splicing. The mechanism is predicted to be catalyzed by the small nuclear RNA (snRNA) components of spliceosomes. To obtain new tertiary constraints between the RNA components, we produced and mapped crosslinks between U6 snRNA and the proximal RNAs of active yeast spliceosomes ("yeast" in this report is Saccharomyces cerevisiae). Thus, specific sites in U6, when substituted with a photoreactive 4-thiouridine or 5-iodouridine, produced spliceosome-dependent crosslinks to U2 snRNA, or in one case, to the pre-mRNA substrate. One set of U2-U6 crosslinks formed before the Prp2p-dependent step of spliceosome assembly, whereas another set formed during or after this step but before the first chemical step of splicing. This latter set of crosslinks formed across U2-U6 helix I. Importantly, this set provides new tertiary constraints for developing 3D models of fully assembled yeast spliceosomes, which are poised for the first chemical step of splicing.

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“…comm.) of the original method (Fabrizio et al 1989) as follows: Extracts were incubated under splicing conditions for 30 min at 30 C in the presence of 1 mM d1 oligonucleotide (CTGTATTGTTTCAAA). Anti-d1 oligonucleotide (TTTGAAA CAATACAG) was added (final concentration 1 mM), and the reaction was incubated at 30 C for 5 min before addition of sitespecifically labeled U6 snRNA (final concentration 1 nM).…”

Section: Rna Interactions Of Splicing Factor Prp8mentioning

confidence: 99%

Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome

Turner¹,

Norman²,

Churcher³

et al. 2006

RNA

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Current models of the core of the spliceosome include a network of RNA-RNA interactions involving the pre-mRNA and the U2, U5, and U6 snRNAs. The essential spliceosomal protein Prp8 interacts with U5 and U6 snRNAs and with specific pre-mRNA sequences that participate in catalysis. This close association with crucial RNA sequences, together with extensive genetic evidence, suggests that Prp8 could directly affect the function of the catalytic core, perhaps acting as a splicing cofactor. However, the sequence of Prp8 is almost entirely novel, and it offers few clues to the molecular basis of Prp8-RNA interactions. We have used an innovative transposon-based strategy to establish that catalytic core RNAs make multiple contacts in the central region of Prp8, underscoring the intimate relationship between this protein and the catalytic center of the spliceosome. Our analysis of RNA interactions identifies a discrete, highly conserved region of Prp8 as a prime candidate for the role of cofactor for the spliceosome's RNA core.

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In vitro assembly of yeast U6 snRNP: a functional assay.

Cited by 130 publications

References 54 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

New tertiary constraints between the RNA components of active yeast spliceosomes: A photo-crosslinking study

Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome

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