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. 2011 Mar 11;331(6022):1289-95.
doi: 10.1126/science.1198830.

Ordered and dynamic assembly of single spliceosomes

Aaron A Hoskins  1 , Larry J Friedman, Sarah S Gallagher, Daniel J Crawford, Eric G Anderson, Richard Wombacher, Nicholas Ramirez, Virginia W Cornish, Jeff Gelles, Melissa J Moore
Affiliations

Ordered and dynamic assembly of single spliceosomes

Aaron A Hoskins et al. Science. .

Abstract

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

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Figures

Figure 1
Figure 1
Preparation and analysis of fluorescently-labeled spliceosome subcomplexes by CoSMoS.
Figure 2
Figure 2
Individual DHFR-labeled subcomplexes binding to surface-tethered pre-mRNAs. Ex. = excitation wavelength; Em. = emission wavelength. (A–C) Images of three fields of view (FOV, 20 × 20 µm), each at two different emission wavelengths, showing that U1 DHFR Cy3-TMP fluorescence signals (spots) are only observed when wt pre-mRNA is present (C). (D) U1 spots versus time in individual FOVs containing RNAs indicated. Note: For wt pre-mRNA, the decrease in spot number after 10 minutes is due to Cy3-TMP photobleaching in this experiment. Experiments in (A–D) contained ATP. (E–F) Smoothed (9-point moving block averaged) curves of indicated subcomplex spots per pre-mRNA versus time, minus (E) or plus (F) ATP. Each subcomplex was monitored in a different WCE. Data in (F) are the average of N = 4 replicates.
Figure 3
Figure 3
(A) Scheme for detecting subcomplex binding to and splicing of the same pre-mRNA molecule. (B and C) Example single molecule traces of U1-SNAP (B) and NTC-SNAP (C) binding to pre-mRNAs (red boxes in insets) that had Alexa647 fluorescence at t = 0 (left insets), but not at t = 60 (right insets).
Figure 4
Figure 4
(A and B) Images of two FOVs taken at three different wavelengths with triple label extract to monitor U1-DHFR/Cy5-TMP and U2-SNAP-DY549 association with Alexa488-labeled pre-mRNA, without (A) or with (B) ATP. (C) Magnification of dashed area in (B) showing co-localization of U1 (yellow boxes) with U2 (white spots). (D) Fluorescence intensity traces showing association of U1 and U2 with an individual pre-mRNA molecule (not shown) in the presence of ATP. Arrival times for each subcomplex (tU1 and tU2) are marked. (E) Histogram of the delay between subcomplex arrival times (tU2-tU1). (F, H) Single molecule fluorescence intensity traces for U2/U5 (F) and U5/NTC (H) bound to single pre-mRNA molecules (not shown). (G, I) Histograms of the delays between U2 and U5 binding (G) and U5 and NTC binding (I).
Figure 5
Figure 5
(A–D) Single molecule traces of SNAP-DY549 labeled subcomplexes binding and dissociating multiple times from individual pre-mRNA molecules (not shown) in the presence of ATP. Arrows indicate durations of two U1 binding events (dwell times) used to analyze subcomplex lifetimes. (E) Kinetic scheme for spliceosome assembly and splicing of RP51A pre-mRNA. Our results provide evidence for reversible binding of all of the major subcomplexes (backward arrows), while others have provided evidence for reversibility of splicing chemistry (8). There is as yet no evidence for reversibility of the activation step prior to splicing or mRNA release.
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