New insights into the spliceosome by single molecule fluorescence microscopy
Highlights
► Pre-mRNA splicing is carried out by a MegaDalton machine called the spliceosome. ► Single molecule fluorescence combined with chemical biology techniques has recently enabled real time visualization of splicing and spliceosome assembly. ► Single molecule FRET measurements reveal pre-mRNA dynamics during splicing. ► Monitoring the assembly of spliceosomes on single pre-mRNAs exposes both ordering and reversibility of key steps.
Introduction
Removal of introns from nascent RNA transcripts (precursors to messenger RNAs or pre-mRNAs) is an essential step in eukaryotic gene expression. The enzyme responsible for this process is the spliceosome, which carries out intron excision via two energy-neutral transesterification reactions: lariat intron formation and exon ligation. Despite the straightforward nature of the chemistry, the spliceosome itself is an extraordinarily complex, 2–3 MDa machine composed of 5 uridine-rich small nuclear RNAs (U1, U2, U4, U5 and U6 snRNAs) and anywhere from 90 to >300 proteins [1, 2]. The snRNAs and a subset of the proteins form stable particles called small nuclear ribonucleoproteins (snRNPs) that constitute the largest building blocks of the spliceosome. Aided by a plethora of more loosely associated proteins (‘splicing factors’), the snRNPs interact with one another and the pre-mRNA to complete each round of splicing via an extraordinarily dynamic process. The current model for spliceosome assembly involves step-wise association of first U1, then U2, followed by U4/U6.U5 tri-snRNP and the multi-protein Prp19 complex (NTC). Once all the major pieces are in place, additional structural rearrangements lead to U1 and U4 expulsion, catalytic activation, lariat formation, exon ligation, spliced product release and finally dissociation of the remaining components (Figure 1). Amazingly, this entire sequence is believed to occur anew on every intron in each pre-mRNA molecule, rendering each assembled and catalytically activated spliceosome a single-turnover enzyme.
As has been recently reviewed [3, 4, 5•, 6], our current understanding of spliceosome assembly is based largely on the procession of stable complexes that form upon addition of a simplified splicing substrate (i.e. two short exons separated by an efficiently spliced intron) to an in vitro splicing reaction. Spliceosomal components are most commonly provided as an Saccharomyces cerevisiae whole cell extract (WCE) or as a mammalian nuclear cell extract (NCE). Stable complexes are resolved by native polyacrylamide gel electrophoresis (PAGE) or by density gradient centrifugation and can be purified by affinity chromatography. In some cases, such purified complexes retain the ability to carry out an individual step in the overall process (see, e.g. [7, 8]). Combined with more than two decades of intensive genetic dissection of the yeast spliceosome, these biochemical studies have yielded tremendous insight into the overall composition, structure and operation of the splicesosome. Nonetheless, what was missing until recently was any detailed kinetic information about the comings and goings of individual components and related structural transitions.
At any single moment in a splicing reaction, different spliceosomes are in different states and doing different things. Averaging of these states and behaviors across an entire population of molecules leads to significant loss of information about molecular dynamics. A means around this is to monitor the behavior of each spliceosome individually. Single molecule approaches can yield a wealth of information about the stochastic and kinetic behaviors of biological systems, their static and dynamic heterogeneities, and the rates of individual steps in multi-step processes. They can also reveal rare and transient species along a reaction pathway that are difficult to detect in ensemble experiments [9, 10]. This review focuses on recent advances in the application of single molecule fluorescence microscopy to the pre-mRNA splicing machinery and the novel insights derived from these analyses. Key to the success of these studies is chemical biology approaches for introducing bright fluorophores into nucleic acids and proteins.
Section snippets
Watching introns leave
Splicing of pre-mRNAs in vitro occurs over tens of minutes. Sustained microscope observation of individual molecules on this time scale requires their immobilization on a surface to prevent diffusion out of the field of view (Figure 2). The most common surface attachment method is a biotin:streptavidin:biotin sandwich, with one biotin molecule being linked to the glass surface (e.g. through a biotinylated polyethylene glycol chain) and the second biotin attached covalently to the pre-mRNA or to
RNAs moving about
It has long been appreciated that pre-mRNA conformational changes are a pre-requisite for splicing: minimally the last nucleotide of the 5′ exon must first be juxtaposed with the branch site for lariat formation and subsequently with the 3′SS for exon ligation (Figure 1). While the full extent of conformational changes necessary for transition between the first and second catalytic steps of splicing is not known, there is ample evidence that the spliceosomal active site is flexible with
Seeing RNA and protein complexes coming and going
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 (Figure 1). 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
Future prospects
Through the combined efforts of several laboratories, single molecule splicers now have the tools in hand to monitor conformational changes in pre-mRNAs or snRNAs during splicing by SM-FRET and the association and dissociation of spliceosome components by CoSMoS. The combination of these approaches (FRET-CoSMoS) to correlate specific structural transitions during splicing with the presence or absence of a given spliceosomal component is likely to represent the next major advance in single
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by NIH National Research Service Award fellowship GM079971 (A.A.H.), K99/R00 GM086471 (A.A.H.), and RO1s GM043369 (J.G.), GM81648 (J.G.), and GM053007 (M.J.M). M.J.M. is a Howard Hughes Medical Institute investigator.
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Present address: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706, USA.