Conformational dynamics of single pre-mRNA molecules during in vitro splicing

Abstract

The spliceosome is a complex small nuclear RNA (snRNA)-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single-molecule FRET (smFRET) that uses a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5' and 3' splice sites. During splicing in vitro, we observed a multitude of generally reversible time- and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3'-splice site mutants differ from one another and from wild type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.

Figure 1

Canonical spliceosome assembly pathway.

Figure 2

Splicing activity of different Ubc4 pre–mRNA variants. (a) Positioning the dyes in different sites in the 20–nt exon of Ubc4 pre–mRNA results in similar splicing efficiency. In this experiment splicing was assayed at room temperature as described in Methods. The three lanes for each pre–mRNA are (−)ATP, (+)ATP for 15 min and (+)ATP for 30 min. All pre–mRNAs examined have an apparent splicing efficiency of first plus second step [(mRNA+lariat intermediate)/(pre–mRNA+ mRNA+lariat intermediate)] of between 30–40%. The Cy5 fluorescence scan is shown. (b) The splicing assay of wildtype (WT), 3′ splice site mutant (3′SS) and branchpoint mutant (BP) Ubc4 pre–mRNAs shows that the 3′SS mutant RNA only carries out step 1, leading to a lariat–intron intermediate. The BP mutant is inactive in splicing. The experiment was carried out as described above for panel A. The Cy5 fluorescence scan is shown. (c) Design of an in situ assay to probe for the presence of introns in immobilized (pre–)mRNAs. (d) Results of RNase H probing for the presence of the intron after incubation of WT, BP, and 3′SS pre–mRNA in yeast splicing extract. Control experiments on WT pre–mRNA and mature mRNA (leftmost 4 columns) show that an intron containing substrate strongly loses Cy3 signal after incubation with a complementary DNA oligonucleotide and RNase H, but not the intron–free mRNA. Loss of the intron to splicing is thus indicated by a small difference in signal before and after RNase H treatment. Error bars indicate 1 s.d. from the mean.

Figure 3

Data analysis and examples of analysis. (a) Synthetic Ubc4 pre mRNA is hybridized via a 17–nt 2′–O–methyl RNA tether to the 3′ exon, and attached via biotin to a streptavidin coated quartz slide. (b) Raw Cy3 (green), Cy5 (red), FRET (Magenta) time trajectories are analyzed using Hidden Markov Modeling (HMM) algorithms to yield idealized trajectories (black) as described in Methods. (c) The first 10 s of the raw FRET trajectories of a subset of (36 out of 175) WT molecules incubated in ATP–depleted cell extract were analyzed to determine the ensemble distribution of FRET states within the population of molecules analyzed. (d) Transition Density Plots (TDPs) utilize the idealized FRET trajectories to determine the entire set of transitions for a given set of molecules. The number of times a transition occurs is represented as a heat map whose index is defined by the color bar. The fact that this analysis, of a subset (36 of 175 molecules as in panel c) of the data shown in Figs. 5a and 6a, provides qualitatively the same result obtained for the full data set attests to the convergence of the analysis.

Figure 4

Conformational dynamics of wildtype (WT), 3′ splice site mutant (3′SS), and branchpoint mutant (BP) pre–mRNA substrates in splicing buffer. (a) Sample traces of all three substrates in splicing buffer, showing raw donor (Cy3, green), acceptor (Cy5, red), and FRET (blue) trajectories and their idealized HMM models (black). (b) FRET histograms of the three pre–mRNAs in splicing buffer. (c) TDPs for all three pre–mRNAs in splicing buffer.

Figure 5

ATP–dependent conformational dynamics of the wildtype (WT), 3′ splice site mutant (3′SS), and branchpoint mutant (BP) pre–mRNAs in yeast cell extract. (a) Probability distributions of FRET states for the WT substrate for each experimental condition. (b) Probability distributions of FRET states for the 3′SS substrate for each experimental condition. (c) Probability distributions of FRET states for the BP substrate for each experimental condition.

Figure 6

Mapping conformational changes of the wildtype (WT) pre–mRNA during spliceosome assembly and splicing in vitro. (a) TDPs for WT substrate; in ATP–depleted extract, within 15 min after ATP addition to cell extract, and after 60 min incubation in (+)ATP cell extract. (b) Representative trajectories (donor, green; acceptor, red; FRET, magenta) and idealized HMM models (black) for WT substrate.

Figure 7

Mapping conformational changes of the 3′ splice site mutant (3′SS) pre–mRNA spliceosome assembly and splicing in vitro. (a) TDPs for 3′SS substrate; in ATP–depleted extract, within 15 min after ATP addition to cell extract, and after 60 min incubation in (+)ATP cell extract. (b) Representative trajectories (donor, green; acceptor, red; FRET, magenta) and idealized HMM models (black) for 3″SS substrate.

Figure 8

Mapping conformational changes of the branchpoint mutant (BP) pre–mRNA spliceosome assembly and splicing in vitro. (a) TDPs for BP substrate; in ATP–depleted extract, within 15 min after ATP addition to cell extract, and after 60 min incubation in (+)ATP cell extract. (b) Representative trajectories (donor, green; acceptor, red; FRET, magenta) and idealized HMM models (black) for BP substrate.

Figure 9

Detailed comparison of kinetic and conformational profiles of pre–mRNAs during spliceosome assembly and splicing in vitro. (a) POKIT plots for WT substrate; in ATP–depleted extract, within 15 min after ATP addition to cell extract, and after 60 min incubation in (+)ATP cell extract. (b) POKIT plots for 3′SS substrate; in ATP–depleted extract, within 15 min after ATP addition to cell extract, and after 60 min incubation in (+)ATP cell extract. (c) POKIT plots for BP substrate; in ATP–depleted extract, within 15 min after ATP addition to cell extract, and after 60 min incubation in (+)ATP cell extract.