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

Abstract

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...

The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome

“…3). Consistent with this conclusion, recent single-molecule FRET studies indicate that the branch site and 5 ′ splice site move closer to one another at or near the Prp2p stage (Crawford et al 2013;Krishnan et al 2013). Since helix Ia is already formed at the Prp2p stage and the spliceosome is not yet competent to undergo the chemistry of splicing, our data imply that helix Ia-mediated juxtaposition of the 5 ′ splice site and branch site is not sufficient to promote the first step of splicing.…”

Conformational dynamics of stem II of the U2 snRNA

“…Labeled RNA fragments were joined using splinted ligation (Crawford et al 2013) with a DNA splint (AAAAGGTAATGAGCC TCATTGAGGTCATTTCA) and the double-stranded T4 RNA ligase 2 (NEB). Ligated RNAs were purified using a denaturing polyacrylamide gel electrophoresis, eluted from the gel fragments, resuspended in water, and stored as concentrated stocks (1 µM) at −20°C.…”

“…RNAs were immobilized onto PEG/PEG-biotin derivatized quartz slides as previously described (Crawford et al 2013). smFRET experiments were carried out in 1× imaging buffer (20 mM HEPES at pH 7.9, 1 mM EDTA, 125 mM KCl, 5% glycerol, 0.1% Triton X-100, 1 mM DTT) using protocatechuate dioxygenase as the oxygen scavenger.…”

“…smFRET and colocalization experiments have provided ample evidence for reversible steps in both spliceosome assembly (Hoskins et al 2011) and pre-mRNA conformation (Abelson et al 2010;Crawford et al 2013;Krishnan et al 2013). Even during late stages of assembly, the spliceosome-associated pre-mRNA undergoes a number of conformational transitions (Abelson et al 2010;Crawford et al 2013;Krishnan et al 2013).…”

“…Even during late stages of assembly, the spliceosome-associated pre-mRNA undergoes a number of conformational transitions (Abelson et al 2010;Crawford et al 2013;Krishnan et al 2013). Some of these transitions are likely occurring due to active site remodeling or "toggling" of the active site between different structures.…”