In addition to understanding conformation changes in the RNAs, it is necessary to define the pathway(s) by which spliceosome composition changes over the course of the splicing process (). Protein inventories of isolated splicing complexes have revealed that dozens of proteins may specifically associate with or dissociate from the spliceosome during transitions between stable intermediates [1
]. While ensemble analyses had suggested that spliceosome assembly occurs by stepwise addition and removal of components [26
], it was not previously known to what extent these reactions are ordered on individual pre-mRNA molecules or if other pathways (e.g. pre-association of the snRNPs [29
]) contribute to spliceosome formation on a subset of molecules. A recent study combining chemical biology approaches for fluorescently labeling spliceosomal subcomplexes with multiwavelength total internal reflectance fluorescence (TIRF) microscopy shed new light on the order and dynamics of spliceosome assembly [30
Hoskins et al. [30
] employed homologous recombination to create haploid yeast strains containing C-terminal protein tags on a variety of essential spliceosomal proteins. The protein tags were comprised of a short glycine/serine linker followed by either the E. coli
dihydrofolate reductase (DHFR) enzyme [31
] or the SNAP tag, a variant of human alkylguanine S
]. After preparation of yeast WCE, DHFR tags were labeled by addition of Cy3- or Cy5-trimethoprim (TMP) analogs to the WCE. Although TMP binds prokaryotic DHFRs non-covalently, the interaction is extremely tight (KD
< 1 nM) [33
]. The SNAP tag was labeled by incubation of WCE with fluorescent benzyl-guanine derivatives [e.g., Snap Surface 549™ (DY549), New England Biolabs] that covalently modify the active-site cysteine of the SNAP tag; excess dye was then removed by gel filtration. Using both the DHFR and SNAP tags in a single yeast strain enabled the authors to label two spliceosomal subcomplexes with spectrally distinguishable fluorophores in the same WCE (e.g., a U1-DHFR/Cy5-TMP-labeled, U2-SNAP/DY549-labeled WCE). Colocalization of the fluorescent subcomplexes on surface-tethered pre-mRNAs was monitored by Colocalization Single Molecule Spectroscopy (CoSMoS). These experiments were enabled by a novel TIRF optical system that efficiently collects fluorescence from multiple fluorophores simultaneously [34
Using CoSMoS to simultaneously monitor association of pairs of spliceosomal subcomplexes (U1/U2, U2/U5, or U5/NTC) with pre-mRNAs in real-time (), Hoskins et al. found that spliceosome assembly occurred stepwise (U1->U2->U5->NTC) on >90% of the pre-mRNA molecules examined. Further, comparison of the rates of spliceosome subcomplex association revealed that no kinetic bottleneck was present for any single subcomplex association. Thus, spliceosome assembly is a kinetically efficient process in vitro.
Another key finding of the Hoskins et al. study was the reversibility of every major assembly step. Inspection of hundreds of single molecule traces revealed that every spliceosomal subcomplex bound dynamically. These results have important implications for the concept of splicing commitment. That is, at what stage of the assembly process does the splicing machinery commit to the utilization of a particular pair of splice sites? Prior to the Hoskins et al. study, it was generally believed that commitment occurs at the earliest stage of assembly – U1 addition [35
]. However, by comparing intron loss events between early (U1) and late (NTC) binding subcomplexes, Hoskins et al. showed that commitment of individual pre-mRNAs is not an all-or-nothing event at the beginning. Because U1 binding is highly dynamic, a given interaction with U1 does not strongly commit a pre-mRNA to intron excision. Rather, the degree of commitment increases as the spliceosome assembles. In both early and late stages of assembly, reversible subcomplex binding allows spliceosomes to disassemble from the pre-mRNA without splicing. These results may have significant implications for our understanding of the regulation of alternative splicing. If spliceosomes formed around a particular set of splice sites can disassemble at any stage prior to intron removal, regulation of splice site choice could occur at any point in the pathway rather than just at the beginning. Thus regulation of alternative splicing is likely to be even more complicated than currently envisioned.