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Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing

Nature Structural & Molecular Biology volume 20, pages 1450–1457 (2013)Cite this article

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Abstract

The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns during the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used single-molecule fluorescence resonance energy transfer to monitor the distance of the 5′ splice site (5′ SS) and branch point (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately before the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5′ SS and BP that accommodate chemistry. Addition of Cwc25, a small heat-stable splicing factor, then strongly biases this equilibrium toward the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor 'pawl', a principle possibly widespread among the many helicase-driven RNPs.

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Figure 1: The SiMPull-FRET approach used to interrogate active splicing complexes.
Figure 2: In the Prp2-stalled Bact complex, the pre-mRNA is restricted predominantly to a static low-FRET state.
Figure 3: Upon the addition of ATP, Prp2 and Spp2, the pre-mRNA is able to explore splice-site proximity.
Figure 4: Under C complex conditions, the pre-mRNA accesses dynamic and stabilized high-FRET states.
Figure 5: Cwc25 enhances the first step of splicing by stabilizing the H state.
Figure 6: Prp2-mediated spliceosome remodeling creates a binding site for Cwc25 near the BP.
Figure 7: Model for the conformational mechanism of first-step splicing.

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Acknowledgements

The authors wish to thank R. Lührmann (Max Planck Institute for Biophysical Chemistry) and R.J. Lin (Fujian Medical University) for generously supplying expression plasmids for Spp2, Cwc25 and Prp2; H. Hadjivassiliou and A. Price (University of California, San Francisco) for providing Cy5–body labeled actin pre-mRNA substrate; D. Semlow and J.P. Staley (University of Chicago) for providing the dominant-negative Prp16 protein. The authors acknowledge funding through US National Institutes of Health grants R01GM098023 to N.G.W. and J.A. and R01GM021119 to C.G.

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Authors and Affiliations

  1. Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan, USA

    Ramya Krishnan, Mario R Blanco, Matthew L Kahlscheuer & Nils G Walter

  2. Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan, USA

    Mario R Blanco

  3. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA

    John Abelson & Christine Guthrie

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  1. Ramya Krishnan
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Contributions

R.K., M.R.B. and M.L.K. performed the biochemical and single-molecule experiments and performed data analysis. M.L.K. cloned, expressed and labeled the proteins. R.K., M.R.B., M.L.K., J.A., C.G. and N.G.W. wrote the manuscript.

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Correspondence to Nils G Walter.

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Integrated supplementary information

Supplementary Figure 1 Binding specificity of Bact complex and purified proteins used for reconstitution.

(a) Field of view showing direct binding of the 5' biotinylated pre-mRNA to the streptavidin on the slide surface saturated with biotin-IgG and free biotin. (b) Field of view showing the binding of the immunopurified Bact spliceosome (with Cef1-TAP) to the streptavidin on the slide surface saturated with free biotin and biotin-IgG. (c) Field of view showing the binding of the immunopurified Bact spliceosome (with Cef1-TAP) to the streptavidin on the slide surface saturated with free biotin in the absence of IgG-biotin. Left and right, the Cy3 and Cy5 channels, respectively. A 532-nm and 635-nm laser was used for excitation under all conditions. (d) Quantification of number of molecules under conditions a–c. (e) Protein expression and purification confirmed by SDS-PAGE analysis. Histidine-tagged Cwc25, Prp2 and Spp2 are shown in lanes 1, 2 and 3, respectively. Cy5-fluorophore labeled single-cysteine Cwc25 is shown in lane 4.

Supplementary Figure 2 Confirmation of Bact complex specificity and activity.

(a) 15% Urea-polyacrylamide gel scanned with a variable mode Typhoon imager. The intron and intron-lariat products are observed in the Cy3 scan (left) and the mature mRNA product is visualized in the Cy5 scan (right). Lanes 1, 2 and 3 represent fractions wash, unbound and bound, respectively. Conditions a and c represent wild-type Ubc4 pre-mRNA assembled in Bact complex and immobilized on magnetic beads with biotin-IgG. Condition b is wild-type pre-mRNA assembled in the absence of extract. Bound molecules were reconstituted with or without Micrococcal Nuclease (MNase) treated extract. (b) 6% Urea-polyacrylamide gel scanned using a variable mode Typhoon imager. Affinity-purified Bact complex formed with Cy5-actin pre-mRNA supplemented with Prp2, Spp2 and 2 mM ATP (lane 1) and Prp2, Spp2 and Cwc25 (lane 2). (c) Quantification of lanes 1 and 2 from b.

Supplementary Figure 3 Single molecule clustering and cross-correlation analysis.

(a) Histogram of HMM-idealized states for each of the K-means derived clusters L1, L2, M and H obtained by clustering single molecule trajectories from all the experimental conditions (Bact, B* and C). (b) Representative molecule showing the FRET states assigned by HMM upon K-means clustering. (c) The number of states for clustering was selected through the use of the Bayesian information criterion (BIC). (d) The mean and standard deviation of the FRET states from the four K-means derived clusters is shown. *** indicates an extremely significant (P < 0.001) difference between all pairwise comparisons as determined by the Tukey test. (e) Sample static trajectory from the Bactcondition with the raw donor (Cy3, green), acceptor (Cy5, red) and FRET (black) trajectories, and idealized HMM models (cyan). The corresponding cross correlation analysis of donor and acceptor trajectories with time lags from 0–50 is shown to the right of each panel. (f) Sample dynamic trajectory from the Bactcondition and corresponding cross correlation analysis.

Supplementary Figure 4 Post–first-step splicing signature and single-molecule kinetic analysis.

(a) FRET probability distribution for molecules stalled by the addition of Prp16 (K379A) dominant negative (DN) mutant. (b) TODP for Prp16DN mutant. (c) Transition density plots (TDPs) for the B* and C complex molecules scaled to the number of transitions determined by HMM. (d) Cumulative distribution plot of dwell times extracted for the indicated transition and fit with either a single- or double-exponential rate equation. (e) Parameters for the double-exponential equations fitted to the dwell-time data. To reduce the dimensionality of the data, a weighted average rate constant kw was calculated by utilizing the amplitudes associated with each time constant as weighting factors. kw was used for Keq calculations and rate comparisons between B* and C complex conditions.

Supplementary Figure 5 Confirmation of Bact complex activity using recombinant proteins.

15% polyacrylamide gel scanned with a variable mode Typhoon imager. Ubc4 pre-mRNA assembled in Bact complexes and supplemented with or without recombinant proteins Prp2, Spp2 and Cwc25. This represents the uncropped unedited form of the gel presented in Figure 1.

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Krishnan, R., Blanco, M., Kahlscheuer, M. et al. Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat Struct Mol Biol 20, 1450–1457 (2013). https://doi.org/10.1038/nsmb.2704

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