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Single-molecule fluorescence-based studies on the dynamics, assembly and catalytic mechanism of the spliceosome Chandani Warnasooriya* and David Rueda*1 *Department of Medicine, Section of Virology and Single Molecule Imaging Group, MRC Clinical Centre, Imperial College London, London W12 0NN, U.K.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract Pre-mRNA (precursor mRNA) splicing is a key step in cellular gene expression where introns are excised and exons are ligated together to produce mature mRNA. This process is catalysed by the spliceosome, which consists of five snRNPs (small nuclear ribonucleoprotein particles) and numerous protein factors. Assembly of these snRNPs and associated proteins is a highly dynamic process, making it challenging to study the conformational rearrangements and spliceosome assembly kinetics in bulk studies. In the present review, we discuss recent studies utilizing techniques based on single-molecule detection that have helped overcome this challenge. These studies focus on the assembly dynamics and splicing kinetics in real-time, which help understanding of spliceosomal assembly and catalysis.
Introduction Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA (precursor mRNA) to form mature mRNA [1–3]. It involves excision of introns from nuclear pre-mRNA, the non-protein coding regions, and ligation of exons, the protein-coding regions, to produce mature mRNA [1]. Splicing provides a mechanism for maintaining the mRNA level, which helps cells regulate gene expression [4]. In addition, an alternative splicing plays an important role in cellular protein diversity by forming diverse mature mRNAs from a single pre-mRNA [5–8]. Splicing is catalysed by the spliceosome, a multi-megadalton ribonucleoprotein complex [1] consisting of five snRNPs (small nuclear ribonucleoproteins): U1, U2, U4, U5 and U6 [1,2]. Each snRNP consists of an snRNA (small nuclear RNA), several snRNP-specific proteins, and a common set of proteins called Sm and Lsm proteins [2,9– 11]. Other than the snRNPs, the spliceosome also associates with several other proteins that optimize its catalytic activity [2,9,10]. These snRNPs along with their associated protein factors follow a highly ordered and stepwise pathway during spliceosomal assembly and catalysis, which is conserved among eukaryotes [2,12]. The dynamic nature of spliceosomal components during assembly and catalysis makes it challenging to study the conformations of these components in traditional bulk studies, which makes singlemolecule methods particularly powerful [13,14]. SingleKey words: co-localization single-molecule spectroscopy (CoSMoS), dynamics, fluorescence resonance energy transfer (FRET), pre-mRNA splicing, single-molecule, fluorescence, splicessomal RNA. Abbreviations: BS, branch site; CoSMoS, co-localization single-molecule spectroscopy; Cy3, indocarbocyanine; Cy5, indodicarbocyanine; ISL, intramolecular stem–loop; NTC, nineteen complex; pre-mRNA, precursor mRNA; sm-FRET, single-molecule FRET; snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; SS, splice site. 1 To whom correspondence should be addressed (email [email protected]).
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molecule fluorescence microscopy allows detection of interactions between molecules and their conformational changes one molecule at a time. Therefore this technique provides access to crucial information on how individual molecules or complexes behave in bulk solution, revealing the underlying structural dynamics and heterogeneity in the system. In addition, single-molecule fluorescence microscopy enables identification and characterization of key transient intermediate states that cannot be observed in ensembleaveraged bulk experiments [14–17]. In the present review, we discuss some recent findings in spliceosomal dynamics, assembly and catalysis using single-molecule fluorescence techniques.
Structure and conformational dynamics of the core snRNAs Although splicing assembly requires all five snRNPs, only U2, U5 and U6 have been shown to be present in catalytically active spliceosomes [1,2]. Of these three, U2 and U6 snRNAs are thought to form the catalytic core of the spliceosome [18–20]. These two snRNAs form an extended base-pairing network that serves as an interaction platform for the 5 -SS (splice site) and the BS (branch site) adenosine, which are the substrates of the first step of splicing (Figures 1a and 1b). The complex also contains highly conserved regions (from yeast to human) of the spliceosome, such as the metal-ion-binding U80, the catalytic AGC triad, and the ACAGAGA loop (Figure 1a). Studies with protein-free U2–U6 complexes have shown two-step splicing-related catalysis in vitro [21,22]. It has also been shown that specific regions of U5 snRNA are functionally dispensable from both steps of splicing [23,24]. There are also many similarities between the spliceosome and group II intron RNA-based catalysis. Both enzymes use two trans-esterification reactions for splicing, and require C The
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Figure 1 The spliceosomal U2–U6 snRNA complex adopts multiple conformations in dynamic equilibrium Secondary structure model of the (a) three-helix junction with an intron (grey) bound and (b) four-helix junction. U2 and U6 are shown in blue and purple respectively. The BS adenosine is circled and exons are shown in brown. Highly conserved regions of U6 snRNA are highlighted in red. (c) A schematic diagram of sm-FRET to monitor U2–U6 folding dynamics. The U6 strand is labelled with Cy3 (D), Cy5 (A) and 3 -biotin for surface-immobilization (top panel). The pre-annealed U2–U6 complex is surface-immobilized using biotin–streptavidin. The sm-FRET time trajectory for a single U2–U6 complex at 10 mM Mg2 + (bottom left-hand panel) and the corresponding FRET histogram (bottom right-hand panel), which reveal three distinct FRET states, is shown. (d) U6 mutations, resulting time trajectories and corresponding FRET histograms. Top panels: 6-fold mutant of U6 snRNA, which destabilizes the low FRET (three-helix) conformation. Bottom panel: A91G mutant, which stabilizes the low FRET conformation. (e) Schematic representation of U2 stem I with post-transcriptional modifications (purple) and (f) the resulting time trajectories and corresponding FRET histogram, show similarities between the yeast and human construct. Adapted with permission from [15,33].
at least two catalytic metal ions [25–27]. They also share conserved regions and structural similarities such as an ISL (intramolecular stem–loop), the metal-ion-binding bulge and the AGC catalytic triad within the catalytically important domains [28,29]. The structure of the U2–U6 complex has been a matter of debate for many years. According to early genetic studies in yeast and recent NMR studies [30,31], the AGC triad of U6 base pairs with a region in U2 forming a three-helix junction consisting of helix Ia, Ib, II and III (Figure 1a). In contrast, early mammalian genetic studies and more recent NMR studies have proposed a four-helix junction C The
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structure [18–20,32], in which the AGC triad base pairs with U6 forming an extended ISL (Figure 1b). These seemingly contradictory observations have been partially reconciled in recent sm-FRET (single-molecule FRET) studies showing that a minimal protein-free U2–U6 complex can adopt at least three distinct conformations in dynamic equilibrium, and that their relative stability depends on metal ion concentration (Figure 1c) [15,33]. Several mutations in U6 snRNA helped assign these FRET states to proposed secondary structures. For example, flipping the AGC triad base pairs (Figure 1d), which prevents the formation of helix Ib, hinders the formation of the low FRET conformation. Conversely, an
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A91G mutation in U6 (Figure 1d), which stabilizes helix II and helix Ib, favours the low FRET conformation. On the basis of these results, the authors assigned the low FRET conformation to the three-helix junction structure and the high and intermediate FRET conformations to the fourhelix junction structure. Additional mutations of the highly conserved U80 led to the proposal that the high FRET conformation is stabilized by tertiary interactions that may involve a network of base triples comprising the ACAGAGA loop, U80 and the AGC triad, similar to those found in crystal structures of a Group II intron [29,34,35]. These interactions had been previously postulated by Pyle and others based on those structures [29,36,37]. An interesting aspect of the high FRET conformation is that it brings all the key residues likely to be involved in catalysing the first step of splicing within close proximity. The third intermediate FRET conformation was proposed to correspond to an obligatory folding intermediate containing the extended ISL but lacking the network of base triples. More recent sm-FRET studies on a protein-free human U2–U6 complex revealed structural and dynamic similarities between the yeast and human constructs, further supporting the conformational assignments [33]. Despite the high sequence conservation between yeast and human U2 and U6 snRNAs, the latter carry numerous post-transcriptional modifications, some of which have been proposed to stabilize stem I of U2 [32]. This hypothesis was directly tested using a U2 strand post-transcriptionally modified at stem I (Figure 1e). The data were consistent with stabilization of stem I in U2 by ∼0.5 kcal/mol (1 cal = 4.184 J), which in turn stabilizes the four-helix junction structure over the threehelix structure (Figure 1f). However, the relatively small magnitude of this stabilization led the authors to propose that post-transcriptional modifications in human snRNAs may primarily mediate specific RNA–protein interactions in vivo.
Dynamics of pre-mRNA molecules during splicing Bringing together the BS and the SS must require extensive structural rearrangements in the pre-mRNA during splicing. Recent single-molecule studies have directly monitored these dynamics during spliceosomal assembly and catalysis [38– 41]. Crawford et al. [38] developed an assay to track intron removal with single-molecule resolution in WCE (wholecell extract) and in real-time during splicing of individual pre-mRNA molecules [38]. Using this assay, in combination with colocalization single-molecule spectroscopy (FRETCoSMoS) [40], the authors obtained new insight into the pre-mRNA dynamics during assembly. The proximity of the 5 -SS and the BS adenosine was monitored using a fluorophore-labelled RP51a pre-mRNA (Figure 2a). The study showed that free pre-mRNA samples several FRET states in accordance with the multiple secondary structures the pre-mRNA can adopt. Using labelled spliceosomal subcomplexes [U1, U2, U4/U6.U5 and the NTC (nineteen
complex)] the authors followed spliceosome assembly simultaneously and in real-time [42]. They found that spliceosomal assembly is orderly and reversible, and that pre-mRNA commitment to splicing occurs late in the assembly pathway. More recently the authors showed that U1 snRNP binding stabilizes a low FRET conformation of the pre-mRNA, and that this conformation is maintained throughout the spliceosomal assembly until addition of the NTC (Figure 2b). The resulting FRET data revealed a low FRET state for the pre-mRNA–spliceosome complex just before and just after NTC addition, but higher FRET states were observed after a ∼1 min delay. Overall these results indicate that the later steps in spliceosomal assembly after NTC addition cause this FRET change, suggesting that U1 binding stabilizes a low FRET pre-mRNA conformation in which 5 -SS and BS are far apart from each other. This prevents the occurrence of any chemical reactions between the two sites until the activated spliceosome is ready for splicing. Binding of NTC to the spliceosomal components stabilizes the interactions between U5, U6 and pre-mRNA, and subsequently the assembling spliceosome forms the activated complex to carry out both steps of splicing (Figure 2b). During these steps the 5 -SS and the BS come into close proximity as is evident from the FRET increase. The subsequent loss of the intron and NTC signals indicates the dissociation of spliceosomal components after the completion of exon ligation. Abelson et al. [39,43] also used labelled pre-mRNA at both splice sites and sm-FRET to show that the pre-mRNA undergoes reversible conformational dynamics during splicing. Using a similar approach, Krishnan et al. [41] monitored the distance changes between the 5 -SS and the BS to determine how the binding of spliceosomal activation factors affect pre-mRNA conformation [41]. This study used UBC4 pre-mRNA and yeast splicing extract prepared from a temperature sensitive Prp2-1 allele that can fully assemble but not carry out the first step of splicing [44,45] (Figure 2c). Similar to the results obtained by Crawford et al. [38], the authors showed that premRNA binding to the Bact complex results in several FRET conformations (L1, L2, M and H; Figure 2d), although the majority of molecules appear in a static low FRET conformation (L2). Addition of recombinant Prp2, which facilitates ATP-dependent structural rearrangement in precatalytic spliceosome to form the B* complex, together with its cofactor Spp2 and ATP, results in a shift towards high FRET (H; Figure 2e). In contrast with the predominantly static L2 state observed for Bact , the B* FRET trajectories exhibit dynamic transitions from low FRET states (M and L2) to H. On the basis of these data the authors suggest that B* brings the 5 -SS and BS into close proximity to carry out the first step of splicing. A small population of static molecules in the high FRET state (H) was also observed, which was suggested to correspond to completion of the first step of splicing. To further characterize the static high FRET state, the authors added recombinant Cwc25, which C The
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Figure 2 5 -SS and BS come into close proximity upon spliceosomal activation (a) A schematic diagram of labelled pre-mRNA with Cy3 (donor, D; green) 7-nt upstream of the BS and Cy5/Alexa Fluor® 647 (acceptor, A; red) 7-nt upstream of the 5 -SS. Rectangles, exons; line, intron; triangle, BS adenosine. Wavy lines denote the multiple-atom linkers. (b) A schematic representation of the conformational changes that occur in pre-mRNA during each step of spliceosome assembly and hypothesized FRET values. Same symbols as in (a) for pre-mRNA. Spliceosomal subcomplexes denoted as circles. Pre-mRNA maintains a low FRET conformation until the NTC addition. After the addition of NTC, the pre-mRNA shows a FRET of 0.3. During the first and second steps of splicing the pre-mRNA adopts higher FRET conformations (>0.5). Intron removal and exon ligation result in loss of signal. Reprinted with permission from [40] Daniel J. Crawford, Aaron A. Hoskins, Larry J. Friedman, Jeff Gelles and Melissa J. Moore (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes under stable approach of 5 splice site and branch site, Proceedings c the authors. Published of the National Academy of Sciences of the United States of America, 110(17), pp. 6783–6788 by the National Academy of Sciences. Pre-mRNA undergoes reversible conformational changes before the first step of splicing. (c) Schematic diagram showing the affinity-purified Bact complex immobilized to a streptavidin-coated quartz slide through biotinylated IgG. Pre-mRNA is labelled with Cy3 (green circle) 6 nt downstream of the BS and with Cy5 (red circle) 7 nt upstream of the 5 -SS. (d–f) FRET probability distribution and transition density plots (TDP) for pre-mRNA dynamics at different stages of spliceosomal assembly: (d) at the Bact complex, (e) at the B* complex, and (f) at the C complex. L1, L2, M and H refer to the four states resulting from clustering analysis. (g) Proposed model showing conformational dynamics of pre-mRNA during spliceosomal assembly before the first step of splicing. Pre-mRNA (rectangle, exons; line, intron) labelled with Cy3 (green) and Cy5 (red). In the Bact complex, pre-mRNA arranged into the L2act conformation with low FRET, where
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the 5 -SS and BS are far apart from each other. Binding of Prp2 with Spp2 and ATP hydrolysis facilitates the formation of the B* complex, which exhibits dynamic transitions from low FRET state (L2*) to mid and high FRET conformations, M* and H*. Binding of Cwc25 near the BS of pre-mRNA enhances the formation of the C complex and transition from a mid to high FRET state, which stabilizes the high FRET state Hc . Adapted with permission from [41].
has been proposed to enhance the first step of splicing along with Prp2, Spp2 and ATP. The resulting traces showed that addition of Cwc25 increases the static high FRET population, supporting their assignment (Figure 2f). This result is in agreement with the data by Crawford et al. [38] where they observed a high FRET state until the exons are ligated. Furthermore, by using Cy5 (indodicarbocyanine)-labelled Cwc25 and Cy3 (indocarbocyanine)-labelled pre-mRNA (near BS adenosine), they determined that the binding site of Cwc25 is ∼5 nm from the BS adenosine. Similarly, both studies indicate that until the formation of the catalytically active B* complex, the pre-mRNA adopts a conformation that keeps the 5 -SS and the BS as far apart as possible to prevent premature catalysis. On the basis of the results from this study, the authors also proposed that the dynamic nature of pre-mRNA allows it to sample several conformations until it is ready for catalysis (Figure 2g). After the completion of the first step of splicing, the equilibrium shifts towards the high FRET state, like a classical biased Brownian ratchet working close to thermal equilibrium by the aid of ATPases such as Prp2 (Figure 2g).
Order of the spliceosomal assembly pathway The order of assembly of spliceosomal components has been established for many years on the basis of ensemble averaged experiments: first U1 snRNP binds, followed by U2 snRNPs, which finally recruit the U4/U6.U5 trisnRNP [2]. A recent single-molecule CoSMoS study has tested whether the binding order is the same for all yeast introns. Shcherbakova et al. [46] used eight well-characterized pre-mRNAs with single introns (such as RP51A, ACT1, RPS30A and UBC4) to determine the rate and binding order of spliceosomal subcomplexes to the pre-mRNA [46]. The selected pre-mRNAs were known to have different splicing rates and efficiencies. First they carried out CoSMoS experiments using surface-immobilized labelled pre-mRNA and labelled subcomplexes (U1, U2, U4/U6.U5 and NTC) to measure rates of spliceosomal subcomplex assembly, which they correlate with known splicing kinetics (Figure 3a). The data from this set of experiments show that, despite the variability in splicing kinetics, all pre-mRNAs follow similar assembly rates and form pre-spliceosomes (U1–U2– pre-mRNA complex) before tri-snRNP recruitment. To check the binding order of U1 and U2 on the premRNA, the authors used labelled U1 and U2 with different fluorophores. Interestingly, the data showed that ∼40 % of RPS30A pre-mRNA molecules show binding of U2 before U1. Overall the authors found that all other pre-mRNAs display both U1-first and U2-first pathways with different
probabilities. To test whether both assembly pathways yield splicing-competent complexes, the authors showed that trisnRNP is recruited with similar efficiency and rates (Figures 3b and 3c), and obtained similar loss of intron signal in the single-molecule and ensemble-averaged experiments. The absence of U1 and U2 binding to the mature mRNA suggests that these snRNPs primarily recognize introns and not the exons. Taken together, these studies provide a new insight into the order of spliceosome assembly, suggesting that these alternative pathways may have a role in alternative splicing regulation.
Future prospects The experiments described in the present review utilizing sm-FRET and CoSMoS have revealed important new aspects about spliceosomal dynamics, including the structural dynamics of core snRNAs, the order of spliceosomal assembly and pre-mRNA dynamics [38–42,46], which have shed light on the mechanism of splicing. As for future prospects in this field, studying the dynamics and assembly process in live cells will represent a significant milestone. Since all of these studies have been performed in vitro or with cell extracts, in the future, the focus should shift towards investigating splicing mechanisms in live cells, the physiological environment of the spliceosome. Indeed, some progress is already being made in this direction: splicing in live cells and the dynamics of spliceosomal assembly in vivo have been studied by monitoring labelled pre-mRNAs [47–49]. As shown by Shcherbakova et al. [46], spliceosomes may be assembled following alternative pathways on different pre-mRNAs or even in different cell types. Therefore more studies are required to characterize these pathways in vivo and to elucidate how cells regulate spliceosomal assembly. Such alternative pathways could provide the cell with opportunities to regulate mRNA levels or related cellular processes such as transcription and alternative splicing. In summary, single-molecule approaches provide a powerful approach to measure the molecular dynamics of large complex biological systems by eliminating ensemble averaging. Therefore it is likely that these methods will become essential in the study of the mechanism of spliceosomal assembly and catalysis. Although a number of studies have already provided key insight into the kinetics of splicing, a number of important questions remain unanswered. For example, what are the differences in splicing kinetics among different organisms? What role do protein factors and regulatory motifs play in spliceosomal assembly? How do these factors affect related biological processes such as alternative splicing, and co- and post-transcriptional splicing in cells? Future technical advances in combination C The
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Figure 3 Spliceosome assembly can follow alternative assembly pathways (a) A schematic diagram of the experimental setup. Labelled pre-mRNA (blue fluorophore) is immobilized on to a quartz slide using biotin–streptavidin and the fluorophores were photobleached before the addition of subcomplexes (white star with blue outline). Labelled subcomplexes, U1, U2 and U5, are added to the slide and binding of each complex to the pre-mRNA is detected by red, green and blue lasers respectively. Free subcomplexes in the bulk solution (grey shading) are not detected. (b) Resulting traces for each subcomplex binding. Formation of pre-spliceosome by U1→U2 (red broken line) and U2→U1 (green broken line) pathways and binding of U4/U6.U5 tri-snRNP (blue broken line) are marked. (c) Proposed model showing the spliceosomal assembly via both pathways: U1-first and U2-first does not affect the binding of U4/U6.U5 tri-snRNP. Rectangles, exons; line, intron; circles, subcomplexes. Reproduced from [46]: Inna Shcherbakova, Aaron A. Hoskins, Larry J. Friedman, Victor Serebrov, Ivan R. Correa, ˆ Jr, Ming-Qun Xu, Jeff Gelles and Melissa J. Moore, 2013, Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy, Cell Reports, 20(1), c 2013 The Authors. Published by Elsevier Inc. pp. 151–165
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with emerging microscopy tools, such as super-resolution microscopy, will help address some of these questions.
Acknowledgements We thank K. Chaurasiya and A. Gautam for a critical reading of the paper before submission.
Funding The Rueda laboratory has been supported, in whole or in part, by the National Institutes of Health [grant number GM085116], a CAREER award of the National Science Foundation [grant number MCB-0747285], the Clinical Sciences Center of the Medical Research Council, and a startup grant from Imperial College London.
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40 Crawford, D.J., Hoskins, A.A., Friedman, L.J., Gelles, J. and Moore, M.J. (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5 splice site and branch site. Proc. Natl. Acad. Sci. U.S.A. 110, 6783–6788 CrossRef PubMed 41 Krishnan, R., Blanco, M.R., Kahlscheuer, M.L., Abelson, J., Guthrie, C. and Walter, N.G. (2013) Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat. Struct. Mol. Biol. 20, 1450–1457 CrossRef PubMed 42 Hoskins, A.A., Friedman, L.J., Gallagher, S.S., Crawford, D.J., Anderson, E.G., Wombacher, R., Ramirez, N., Cornish, V.W., Gelles, J. and Moore, M.J. (2011) Ordered and dynamic assembly of single spliceosomes. Science 331, 1289–1295 CrossRef PubMed 43 Abelson, J., Hadjivassiliou, H. and Guthrie, C. (2010) Preparation of fluorescent pre-mRNA substrates for an smFRET study of pre-mRNA splicing in yeast. Methods Enzymol. 472, 31–40 CrossRef PubMed 44 Kim, S.H. and Lin, R.J. (1996) Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol. Cell. Biol. 16, 6810–6819 PubMed 45 Vijayraghavan, U., Company, M. and Abelson, J. (1989) Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 3, 1206–1216 CrossRef PubMed
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46 Shcherbakova, I., Hoskins, A.A., Friedman, L.J., Serebrov, V., Correa, ˆ Jr, I.R., Xu, M.Q., Gelles, J. and Moore, M.J. (2013) Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy. Cell Rep. 5, 151–165 CrossRef PubMed 47 Rino, J., Carvalho, T., Braga, J., Desterro, J.M., Luhrmann, R. and Carmo-Fonseca, M. (2007) A stochastic view of spliceosome assembly and recycling in the nucleus. PLoS Comput. Biol. 3, 2019–2031 CrossRef PubMed 48 Huranova, M., Ivani, I., Benda, A., Poser, I., Brody, Y., Hof, M., Shav-Tal, Y., Neugebauer, K.M. and Stanek, D. (2010) The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J. Cell Biol. 191, 75–86 CrossRef PubMed 49 Martin, R.M., Rino, J., Carvalho, C., Kirchhausen, T. and Carmo-Fonseca, M. (2013) Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity. Cell Rep. 4, 1144–1155 CrossRef PubMed
Received 29 April 2014 doi:10.1042/BST20140105
Single-molecule fluorescence-based studies on the dynamics, assembly and catalytic mechanism of the spliceosome Chandani Warnasooriya* and David Rueda*1 *Department of Medicine, Section of Virology and Single Molecule Imaging Group, MRC Clinical Centre, Imperial College London, London W12 0NN, U.K.
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Abstract Pre-mRNA (precursor mRNA) splicing is a key step in cellular gene expression where introns are excised and exons are ligated together to produce mature mRNA. This process is catalysed by the spliceosome, which consists of five snRNPs (small nuclear ribonucleoprotein particles) and numerous protein factors. Assembly of these snRNPs and associated proteins is a highly dynamic process, making it challenging to study the conformational rearrangements and spliceosome assembly kinetics in bulk studies. In the present review, we discuss recent studies utilizing techniques based on single-molecule detection that have helped overcome this challenge. These studies focus on the assembly dynamics and splicing kinetics in real-time, which help understanding of spliceosomal assembly and catalysis.
Introduction Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA (precursor mRNA) to form mature mRNA [1–3]. It involves excision of introns from nuclear pre-mRNA, the non-protein coding regions, and ligation of exons, the protein-coding regions, to produce mature mRNA [1]. Splicing provides a mechanism for maintaining the mRNA level, which helps cells regulate gene expression [4]. In addition, an alternative splicing plays an important role in cellular protein diversity by forming diverse mature mRNAs from a single pre-mRNA [5–8]. Splicing is catalysed by the spliceosome, a multi-megadalton ribonucleoprotein complex [1] consisting of five snRNPs (small nuclear ribonucleoproteins): U1, U2, U4, U5 and U6 [1,2]. Each snRNP consists of an snRNA (small nuclear RNA), several snRNP-specific proteins, and a common set of proteins called Sm and Lsm proteins [2,9– 11]. Other than the snRNPs, the spliceosome also associates with several other proteins that optimize its catalytic activity [2,9,10]. These snRNPs along with their associated protein factors follow a highly ordered and stepwise pathway during spliceosomal assembly and catalysis, which is conserved among eukaryotes [2,12]. The dynamic nature of spliceosomal components during assembly and catalysis makes it challenging to study the conformations of these components in traditional bulk studies, which makes singlemolecule methods particularly powerful [13,14]. SingleKey words: co-localization single-molecule spectroscopy (CoSMoS), dynamics, fluorescence resonance energy transfer (FRET), pre-mRNA splicing, single-molecule, fluorescence, splicessomal RNA. Abbreviations: BS, branch site; CoSMoS, co-localization single-molecule spectroscopy; Cy3, indocarbocyanine; Cy5, indodicarbocyanine; ISL, intramolecular stem–loop; NTC, nineteen complex; pre-mRNA, precursor mRNA; sm-FRET, single-molecule FRET; snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; SS, splice site. 1 To whom correspondence should be addressed (email [email protected]).
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molecule fluorescence microscopy allows detection of interactions between molecules and their conformational changes one molecule at a time. Therefore this technique provides access to crucial information on how individual molecules or complexes behave in bulk solution, revealing the underlying structural dynamics and heterogeneity in the system. In addition, single-molecule fluorescence microscopy enables identification and characterization of key transient intermediate states that cannot be observed in ensembleaveraged bulk experiments [14–17]. In the present review, we discuss some recent findings in spliceosomal dynamics, assembly and catalysis using single-molecule fluorescence techniques.
Structure and conformational dynamics of the core snRNAs Although splicing assembly requires all five snRNPs, only U2, U5 and U6 have been shown to be present in catalytically active spliceosomes [1,2]. Of these three, U2 and U6 snRNAs are thought to form the catalytic core of the spliceosome [18–20]. These two snRNAs form an extended base-pairing network that serves as an interaction platform for the 5 -SS (splice site) and the BS (branch site) adenosine, which are the substrates of the first step of splicing (Figures 1a and 1b). The complex also contains highly conserved regions (from yeast to human) of the spliceosome, such as the metal-ion-binding U80, the catalytic AGC triad, and the ACAGAGA loop (Figure 1a). Studies with protein-free U2–U6 complexes have shown two-step splicing-related catalysis in vitro [21,22]. It has also been shown that specific regions of U5 snRNA are functionally dispensable from both steps of splicing [23,24]. There are also many similarities between the spliceosome and group II intron RNA-based catalysis. Both enzymes use two trans-esterification reactions for splicing, and require C The
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Figure 1 The spliceosomal U2–U6 snRNA complex adopts multiple conformations in dynamic equilibrium Secondary structure model of the (a) three-helix junction with an intron (grey) bound and (b) four-helix junction. U2 and U6 are shown in blue and purple respectively. The BS adenosine is circled and exons are shown in brown. Highly conserved regions of U6 snRNA are highlighted in red. (c) A schematic diagram of sm-FRET to monitor U2–U6 folding dynamics. The U6 strand is labelled with Cy3 (D), Cy5 (A) and 3 -biotin for surface-immobilization (top panel). The pre-annealed U2–U6 complex is surface-immobilized using biotin–streptavidin. The sm-FRET time trajectory for a single U2–U6 complex at 10 mM Mg2 + (bottom left-hand panel) and the corresponding FRET histogram (bottom right-hand panel), which reveal three distinct FRET states, is shown. (d) U6 mutations, resulting time trajectories and corresponding FRET histograms. Top panels: 6-fold mutant of U6 snRNA, which destabilizes the low FRET (three-helix) conformation. Bottom panel: A91G mutant, which stabilizes the low FRET conformation. (e) Schematic representation of U2 stem I with post-transcriptional modifications (purple) and (f) the resulting time trajectories and corresponding FRET histogram, show similarities between the yeast and human construct. Adapted with permission from [15,33].
at least two catalytic metal ions [25–27]. They also share conserved regions and structural similarities such as an ISL (intramolecular stem–loop), the metal-ion-binding bulge and the AGC catalytic triad within the catalytically important domains [28,29]. The structure of the U2–U6 complex has been a matter of debate for many years. According to early genetic studies in yeast and recent NMR studies [30,31], the AGC triad of U6 base pairs with a region in U2 forming a three-helix junction consisting of helix Ia, Ib, II and III (Figure 1a). In contrast, early mammalian genetic studies and more recent NMR studies have proposed a four-helix junction C The
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structure [18–20,32], in which the AGC triad base pairs with U6 forming an extended ISL (Figure 1b). These seemingly contradictory observations have been partially reconciled in recent sm-FRET (single-molecule FRET) studies showing that a minimal protein-free U2–U6 complex can adopt at least three distinct conformations in dynamic equilibrium, and that their relative stability depends on metal ion concentration (Figure 1c) [15,33]. Several mutations in U6 snRNA helped assign these FRET states to proposed secondary structures. For example, flipping the AGC triad base pairs (Figure 1d), which prevents the formation of helix Ib, hinders the formation of the low FRET conformation. Conversely, an
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A91G mutation in U6 (Figure 1d), which stabilizes helix II and helix Ib, favours the low FRET conformation. On the basis of these results, the authors assigned the low FRET conformation to the three-helix junction structure and the high and intermediate FRET conformations to the fourhelix junction structure. Additional mutations of the highly conserved U80 led to the proposal that the high FRET conformation is stabilized by tertiary interactions that may involve a network of base triples comprising the ACAGAGA loop, U80 and the AGC triad, similar to those found in crystal structures of a Group II intron [29,34,35]. These interactions had been previously postulated by Pyle and others based on those structures [29,36,37]. An interesting aspect of the high FRET conformation is that it brings all the key residues likely to be involved in catalysing the first step of splicing within close proximity. The third intermediate FRET conformation was proposed to correspond to an obligatory folding intermediate containing the extended ISL but lacking the network of base triples. More recent sm-FRET studies on a protein-free human U2–U6 complex revealed structural and dynamic similarities between the yeast and human constructs, further supporting the conformational assignments [33]. Despite the high sequence conservation between yeast and human U2 and U6 snRNAs, the latter carry numerous post-transcriptional modifications, some of which have been proposed to stabilize stem I of U2 [32]. This hypothesis was directly tested using a U2 strand post-transcriptionally modified at stem I (Figure 1e). The data were consistent with stabilization of stem I in U2 by ∼0.5 kcal/mol (1 cal = 4.184 J), which in turn stabilizes the four-helix junction structure over the threehelix structure (Figure 1f). However, the relatively small magnitude of this stabilization led the authors to propose that post-transcriptional modifications in human snRNAs may primarily mediate specific RNA–protein interactions in vivo.
Dynamics of pre-mRNA molecules during splicing Bringing together the BS and the SS must require extensive structural rearrangements in the pre-mRNA during splicing. Recent single-molecule studies have directly monitored these dynamics during spliceosomal assembly and catalysis [38– 41]. Crawford et al. [38] developed an assay to track intron removal with single-molecule resolution in WCE (wholecell extract) and in real-time during splicing of individual pre-mRNA molecules [38]. Using this assay, in combination with colocalization single-molecule spectroscopy (FRETCoSMoS) [40], the authors obtained new insight into the pre-mRNA dynamics during assembly. The proximity of the 5 -SS and the BS adenosine was monitored using a fluorophore-labelled RP51a pre-mRNA (Figure 2a). The study showed that free pre-mRNA samples several FRET states in accordance with the multiple secondary structures the pre-mRNA can adopt. Using labelled spliceosomal subcomplexes [U1, U2, U4/U6.U5 and the NTC (nineteen
complex)] the authors followed spliceosome assembly simultaneously and in real-time [42]. They found that spliceosomal assembly is orderly and reversible, and that pre-mRNA commitment to splicing occurs late in the assembly pathway. More recently the authors showed that U1 snRNP binding stabilizes a low FRET conformation of the pre-mRNA, and that this conformation is maintained throughout the spliceosomal assembly until addition of the NTC (Figure 2b). The resulting FRET data revealed a low FRET state for the pre-mRNA–spliceosome complex just before and just after NTC addition, but higher FRET states were observed after a ∼1 min delay. Overall these results indicate that the later steps in spliceosomal assembly after NTC addition cause this FRET change, suggesting that U1 binding stabilizes a low FRET pre-mRNA conformation in which 5 -SS and BS are far apart from each other. This prevents the occurrence of any chemical reactions between the two sites until the activated spliceosome is ready for splicing. Binding of NTC to the spliceosomal components stabilizes the interactions between U5, U6 and pre-mRNA, and subsequently the assembling spliceosome forms the activated complex to carry out both steps of splicing (Figure 2b). During these steps the 5 -SS and the BS come into close proximity as is evident from the FRET increase. The subsequent loss of the intron and NTC signals indicates the dissociation of spliceosomal components after the completion of exon ligation. Abelson et al. [39,43] also used labelled pre-mRNA at both splice sites and sm-FRET to show that the pre-mRNA undergoes reversible conformational dynamics during splicing. Using a similar approach, Krishnan et al. [41] monitored the distance changes between the 5 -SS and the BS to determine how the binding of spliceosomal activation factors affect pre-mRNA conformation [41]. This study used UBC4 pre-mRNA and yeast splicing extract prepared from a temperature sensitive Prp2-1 allele that can fully assemble but not carry out the first step of splicing [44,45] (Figure 2c). Similar to the results obtained by Crawford et al. [38], the authors showed that premRNA binding to the Bact complex results in several FRET conformations (L1, L2, M and H; Figure 2d), although the majority of molecules appear in a static low FRET conformation (L2). Addition of recombinant Prp2, which facilitates ATP-dependent structural rearrangement in precatalytic spliceosome to form the B* complex, together with its cofactor Spp2 and ATP, results in a shift towards high FRET (H; Figure 2e). In contrast with the predominantly static L2 state observed for Bact , the B* FRET trajectories exhibit dynamic transitions from low FRET states (M and L2) to H. On the basis of these data the authors suggest that B* brings the 5 -SS and BS into close proximity to carry out the first step of splicing. A small population of static molecules in the high FRET state (H) was also observed, which was suggested to correspond to completion of the first step of splicing. To further characterize the static high FRET state, the authors added recombinant Cwc25, which C The
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Figure 2 5 -SS and BS come into close proximity upon spliceosomal activation (a) A schematic diagram of labelled pre-mRNA with Cy3 (donor, D; green) 7-nt upstream of the BS and Cy5/Alexa Fluor® 647 (acceptor, A; red) 7-nt upstream of the 5 -SS. Rectangles, exons; line, intron; triangle, BS adenosine. Wavy lines denote the multiple-atom linkers. (b) A schematic representation of the conformational changes that occur in pre-mRNA during each step of spliceosome assembly and hypothesized FRET values. Same symbols as in (a) for pre-mRNA. Spliceosomal subcomplexes denoted as circles. Pre-mRNA maintains a low FRET conformation until the NTC addition. After the addition of NTC, the pre-mRNA shows a FRET of 0.3. During the first and second steps of splicing the pre-mRNA adopts higher FRET conformations (>0.5). Intron removal and exon ligation result in loss of signal. Reprinted with permission from [40] Daniel J. Crawford, Aaron A. Hoskins, Larry J. Friedman, Jeff Gelles and Melissa J. Moore (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes under stable approach of 5 splice site and branch site, Proceedings c the authors. Published of the National Academy of Sciences of the United States of America, 110(17), pp. 6783–6788 by the National Academy of Sciences. Pre-mRNA undergoes reversible conformational changes before the first step of splicing. (c) Schematic diagram showing the affinity-purified Bact complex immobilized to a streptavidin-coated quartz slide through biotinylated IgG. Pre-mRNA is labelled with Cy3 (green circle) 6 nt downstream of the BS and with Cy5 (red circle) 7 nt upstream of the 5 -SS. (d–f) FRET probability distribution and transition density plots (TDP) for pre-mRNA dynamics at different stages of spliceosomal assembly: (d) at the Bact complex, (e) at the B* complex, and (f) at the C complex. L1, L2, M and H refer to the four states resulting from clustering analysis. (g) Proposed model showing conformational dynamics of pre-mRNA during spliceosomal assembly before the first step of splicing. Pre-mRNA (rectangle, exons; line, intron) labelled with Cy3 (green) and Cy5 (red). In the Bact complex, pre-mRNA arranged into the L2act conformation with low FRET, where
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the 5 -SS and BS are far apart from each other. Binding of Prp2 with Spp2 and ATP hydrolysis facilitates the formation of the B* complex, which exhibits dynamic transitions from low FRET state (L2*) to mid and high FRET conformations, M* and H*. Binding of Cwc25 near the BS of pre-mRNA enhances the formation of the C complex and transition from a mid to high FRET state, which stabilizes the high FRET state Hc . Adapted with permission from [41].
has been proposed to enhance the first step of splicing along with Prp2, Spp2 and ATP. The resulting traces showed that addition of Cwc25 increases the static high FRET population, supporting their assignment (Figure 2f). This result is in agreement with the data by Crawford et al. [38] where they observed a high FRET state until the exons are ligated. Furthermore, by using Cy5 (indodicarbocyanine)-labelled Cwc25 and Cy3 (indocarbocyanine)-labelled pre-mRNA (near BS adenosine), they determined that the binding site of Cwc25 is ∼5 nm from the BS adenosine. Similarly, both studies indicate that until the formation of the catalytically active B* complex, the pre-mRNA adopts a conformation that keeps the 5 -SS and the BS as far apart as possible to prevent premature catalysis. On the basis of the results from this study, the authors also proposed that the dynamic nature of pre-mRNA allows it to sample several conformations until it is ready for catalysis (Figure 2g). After the completion of the first step of splicing, the equilibrium shifts towards the high FRET state, like a classical biased Brownian ratchet working close to thermal equilibrium by the aid of ATPases such as Prp2 (Figure 2g).
Order of the spliceosomal assembly pathway The order of assembly of spliceosomal components has been established for many years on the basis of ensemble averaged experiments: first U1 snRNP binds, followed by U2 snRNPs, which finally recruit the U4/U6.U5 trisnRNP [2]. A recent single-molecule CoSMoS study has tested whether the binding order is the same for all yeast introns. Shcherbakova et al. [46] used eight well-characterized pre-mRNAs with single introns (such as RP51A, ACT1, RPS30A and UBC4) to determine the rate and binding order of spliceosomal subcomplexes to the pre-mRNA [46]. The selected pre-mRNAs were known to have different splicing rates and efficiencies. First they carried out CoSMoS experiments using surface-immobilized labelled pre-mRNA and labelled subcomplexes (U1, U2, U4/U6.U5 and NTC) to measure rates of spliceosomal subcomplex assembly, which they correlate with known splicing kinetics (Figure 3a). The data from this set of experiments show that, despite the variability in splicing kinetics, all pre-mRNAs follow similar assembly rates and form pre-spliceosomes (U1–U2– pre-mRNA complex) before tri-snRNP recruitment. To check the binding order of U1 and U2 on the premRNA, the authors used labelled U1 and U2 with different fluorophores. Interestingly, the data showed that ∼40 % of RPS30A pre-mRNA molecules show binding of U2 before U1. Overall the authors found that all other pre-mRNAs display both U1-first and U2-first pathways with different
probabilities. To test whether both assembly pathways yield splicing-competent complexes, the authors showed that trisnRNP is recruited with similar efficiency and rates (Figures 3b and 3c), and obtained similar loss of intron signal in the single-molecule and ensemble-averaged experiments. The absence of U1 and U2 binding to the mature mRNA suggests that these snRNPs primarily recognize introns and not the exons. Taken together, these studies provide a new insight into the order of spliceosome assembly, suggesting that these alternative pathways may have a role in alternative splicing regulation.
Future prospects The experiments described in the present review utilizing sm-FRET and CoSMoS have revealed important new aspects about spliceosomal dynamics, including the structural dynamics of core snRNAs, the order of spliceosomal assembly and pre-mRNA dynamics [38–42,46], which have shed light on the mechanism of splicing. As for future prospects in this field, studying the dynamics and assembly process in live cells will represent a significant milestone. Since all of these studies have been performed in vitro or with cell extracts, in the future, the focus should shift towards investigating splicing mechanisms in live cells, the physiological environment of the spliceosome. Indeed, some progress is already being made in this direction: splicing in live cells and the dynamics of spliceosomal assembly in vivo have been studied by monitoring labelled pre-mRNAs [47–49]. As shown by Shcherbakova et al. [46], spliceosomes may be assembled following alternative pathways on different pre-mRNAs or even in different cell types. Therefore more studies are required to characterize these pathways in vivo and to elucidate how cells regulate spliceosomal assembly. Such alternative pathways could provide the cell with opportunities to regulate mRNA levels or related cellular processes such as transcription and alternative splicing. In summary, single-molecule approaches provide a powerful approach to measure the molecular dynamics of large complex biological systems by eliminating ensemble averaging. Therefore it is likely that these methods will become essential in the study of the mechanism of spliceosomal assembly and catalysis. Although a number of studies have already provided key insight into the kinetics of splicing, a number of important questions remain unanswered. For example, what are the differences in splicing kinetics among different organisms? What role do protein factors and regulatory motifs play in spliceosomal assembly? How do these factors affect related biological processes such as alternative splicing, and co- and post-transcriptional splicing in cells? Future technical advances in combination C The
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Figure 3 Spliceosome assembly can follow alternative assembly pathways (a) A schematic diagram of the experimental setup. Labelled pre-mRNA (blue fluorophore) is immobilized on to a quartz slide using biotin–streptavidin and the fluorophores were photobleached before the addition of subcomplexes (white star with blue outline). Labelled subcomplexes, U1, U2 and U5, are added to the slide and binding of each complex to the pre-mRNA is detected by red, green and blue lasers respectively. Free subcomplexes in the bulk solution (grey shading) are not detected. (b) Resulting traces for each subcomplex binding. Formation of pre-spliceosome by U1→U2 (red broken line) and U2→U1 (green broken line) pathways and binding of U4/U6.U5 tri-snRNP (blue broken line) are marked. (c) Proposed model showing the spliceosomal assembly via both pathways: U1-first and U2-first does not affect the binding of U4/U6.U5 tri-snRNP. Rectangles, exons; line, intron; circles, subcomplexes. Reproduced from [46]: Inna Shcherbakova, Aaron A. Hoskins, Larry J. Friedman, Victor Serebrov, Ivan R. Correa, ˆ Jr, Ming-Qun Xu, Jeff Gelles and Melissa J. Moore, 2013, Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy, Cell Reports, 20(1), c 2013 The Authors. Published by Elsevier Inc. pp. 151–165
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with emerging microscopy tools, such as super-resolution microscopy, will help address some of these questions.
Acknowledgements We thank K. Chaurasiya and A. Gautam for a critical reading of the paper before submission.
Funding The Rueda laboratory has been supported, in whole or in part, by the National Institutes of Health [grant number GM085116], a CAREER award of the National Science Foundation [grant number MCB-0747285], the Clinical Sciences Center of the Medical Research Council, and a startup grant from Imperial College London.
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Received 29 April 2014 doi:10.1042/BST20140105
