While the experiments in can define the population-averaged timing with which different subcomplexes arrive at the pre-mRNA, they do not directly assess the order of subcomplex addition on individual pre-mRNA molecules. Further, the data in and S5
are composites of subcomplex association and dissociation events, photobleaching, and TMP dye exchange, and are additionally complicated by variations in WCE splicing activity. These issues can be resolved by using CoSMoS to simultaneously follow the pre-mRNA association of two spliceosomal subcomplexes in the same WCE. To do so, we used two DHFR/Cy5-TMP tags and a single SNAP DY549 tag to label two subcomplexes (e.g., U1-DHFR and U2-SNAP) with different fluorophores in the same extract (triple label extracts, ), and visualized them binding to individual Alexa488-labeled pre-mRNA molecules ().
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 (more ...)
As was observed with the individually labeled extracts, when both U1 and U2 were labeled in the same WCE only U1 to co-localized with pre-mRNAs in the absence of ATP, while both U1 and U2 co-localized with pre-mRNAs in the presence of ATP ( and Movie S3
). When individual pre-mRNA molecules were followed over time, the largest class (49%, Table S3
) exhibited at least one discrete onset of U1 fluorescence and at least one discrete onset of U2 fluorescence ( and S7
). Other classes exhibited only U1 binding (18%), only U2 binding (6%), or no binding events (27%). These latter subpopulations may arise from the presence of some nonfluorescent subcomplexes in the extract and/or from alternative conformations of the pre-mRNA (15
) that prevent spliceosome assembly. U1 and U2 spots persisted for seconds to minutes before disappearing due to either dye photobleaching or subcomplex dissociation. For U1, which was labeled with two DHFR tags, fluorescence typically vanished in one or two discrete steps (96% of events, Table S4
). Analogously, for U2, which was labeled with one SNAP tag, fluorescence most often vanished in a single step (88% of events). Thus only one copy each of U1 and U2 is present at any given time on the majority of pre-mRNAs.
To quantitatively evaluate the U1 and U2 binding order on individual pre-mRNA molecules (), we calculated tU2
, the difference between the arrival times of the two subcomplexes (20
). A histogram () shows that the overwhelming majority (90%) of these delay times were positive, indicating that U2 binding nearly always followed U1 binding. This conclusion was confirmed by correlation analysis of the absolute binding times (Figure S8
) which revealed that even U1 binding events occurring late in the experiment were soon followed by U2 binding. While U1 and U2 appeared to arrive simultaneously on a small minority (9 out of 223 events) of pre-mRNAs, some of these are likely cases of U1 and U2 arriving in rapid succession separated by a delay that the experimental time resolution (5–6s) was insufficient to resolve ((20
) and Table S5
). Thus, assembly is highly ordered, with U1 always or almost always binding before U2. Further, >95% of pre-mRNAs that acquire both U1 and U2 acquire them separately rather than as a preformed U1/U2 complex. Consequently, formation of a U1/U2 complex prior to association with pre-mRNA cannot be a requirement for splicing since the fraction of pre-mRNAs that splice is greater than 5% (Table S2
To examine the ordering of later assembly steps, we used the same methodologies with other triply labeled yeast strains. U2 fluorescence almost always preceded onset of U5 fluorescence ( and S9
, Table S6
); 97% of the tU5
values were positive (). Similarly, U5 fluorescence almost always preceded onset of NTC fluorescence ( and S10
, Table S7
); 91% of the tNTC-tU5
delay values were positive (). In both the U2/U5 and U5/NTC data sets, very few traces (Table S5
) exhibited apparent simultaneous binding of the subcomplexes, and analysis of all traces suggested that at most one copy each of U5 and NTC were present on the majority of pre-mRNAs (Table S4
). In sum, our data indicate that when spliceosome assembly is followed on individual RP51A pre-mRNA molecules, the predominant reaction pathway is highly ordered (U1 → U2 → tri-snRNP → NTC). Further, the experiments indicate little or no preassociation for any pair of subcomplexes studied (Table S5
). As with U1/U2, these data demonstrate that no preassociation of these subcomplexes is required for splicing.
On top of providing information about binding order, the CoSMoS methodology permits measurement of defined kinetic parameters. The arrival times of the first U1 subcomplex on each pre-mRNA and all three time-delay data sets (tU2-tU1, tU5-tU2
) are well fit by single exponential distributions (Figure S11
), allowing determination of apparent first-order rate constants (). All four rate constants fall in a narrow range (0.1–0.4 min−1
), suggesting that no single subcomplex association step predominantly limits the rate of spliceosome assembly on RP51A pre-mRNA.
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 (more ...)
In addition to arrival times, the triple label experiments also allowed us to examine the order of subcomplex loss from pre-mRNA. Preliminary analysis revealed that U1 fluorescence tended to be lost before U2 fluorescence, and U2 fluorescence tended to be lost before U5 fluorescence. Only with U5 and NTC did a significant number of pre-mRNAs lose fluorescence from both subcomplexes simultaneously (Table S8
). These results are consistent with known post-assembly events, including ordered loss of U1 and the SF3b component of U2 during spliceosome activation and subsequent simultaneous loss of U5 and NTC coincident with spliced mRNA release (2
). While additional analyses of photobleaching and Cy5-TMP dye exchange rates will be required to fully interpret these results, they do indicate that subcomplex dissociation coupled to activation and spliceosome disassembly is detectable using this methodology. Definitive analysis of subcomplex dissociation relative to catalysis and intron release awaits future development of more photostable splicing reporters.
We also examined dissociation kinetics of each subcomplex (20
). In all cases, good fits of dwell time distributions required a function containing more than one exponential term (Figure S12; Table S9
). This presence of both short-(τ1
< 1 min) and long-lived (τ2
> 1 min) characteristic dwell times indicates that there is more than one species from which each subcomplex can dissociate. Thus, subcomplex dissociation is more complex than some current models suggest, and there are multiple mechanisms consistent with our data (Figure S13
). Elucidation of these mechanisms may be possible by combining CoSMoS with appropriate mutants and inhibitors of assembly.