Many biochemical analyses require well-characterized enzymes and controlled conditions, making ‘purification’ one of the ‘Ten Commandments’ of enzymology [16
]. With such a plethora of components to contend with, obeying this commandment was a seemingly insurmountable hurdle for spliceosome biochemists in the past. Recently, however, advances in affinity purification have made purifying active spliceosomes a reality, and several groups are now employing these methods to great effect.
The Lührmann group has succeeded in purifying fully-assembled and active spliceosomes from both human NE and yeast WCE [17
]. These complexes were first assembled in extracts on pre-mRNAs containing several MS2 phage coat protein recognition RNA hairpins in the 5′ exon. This enabled subsequent spliceosome purification via amylose affinity chromatography with maltose-binding protein (MBP) fused to the MS2 protein. For the human spliceosome, the pre-mRNA substrate contained a 5′ exon and intron, but no 3′ SS or 3′ exon [17
]. Spliceosomes formed on such pre-mRNAs carry out 5′ SS cleavage, but become stalled at the C1 or C2 stage due to absence of a 3′ SS (). It was previously shown that such spliceosomes in unfractionated NE can perform bi-molecular exon ligation when supplied with an additional RNA containing a 3′ SS [19
]. Now it has been shown that when purified, these intermediate-containing spliceosomes can carry out bi-molecular exon ligation without any additional protein factors, suggesting that they are predominantly in the C2 complex form [17
Active yeast spliceosomes were accumulated in WCE from cells carrying a temperature sensitive (Ts) mutation in the Prp2 spliceosomal ATPase [18
]. In heat-inactivated prp2-1
extract, splicing stalls just prior to 5′ exon cleavage at the Bact
] (). Once purified, Bact
was not itself competent for chemistry; 5′ SS cleavage required addition of wild type Prp2, recombinant Spp2 and Cwc25 proteins and ATP. Reconstitution of both 5′ SS cleavage and exon ligation additionally required four second step factors (Prp16, Prp18, Prp22, and Slu7). Thus, transitioning yeast Bact
through both 5′ SS cleavage and exon ligation minimally requires ATP and seven transacting splicing factors. With this system established, it should now be possible to elucidate the exact functions of these seven factors in much greater detail.
Assembly of spliceosomes in extracts allows for incorporation of site-specifically modified pre-mRNAs and snRNAs. For snRNAs, this is accomplished by first depleting the endogenous snRNA via targeted RNaseH cleavage, and then adding back an in vitro
synthesized version containing the modification(s) of interest. The Staley lab recently used this approach to assemble spliceosomes with a synthetic U6 snRNA containing a single phosphorothioate linkage at the pro-Sp
non-bridging oxygen of U80 (). A phosphorothioate at this position had previously been shown to inhibit splicing just prior to 5′ SS cleavage [22
], making it a likely candidate for Mg2+
coordination in the spliceosomal active site. Once assembled, the U6 phosphorothioate spliceosomes were isolated via a tandem affinity purification (TAP) tag [23
] on spliceosomal protein Prp19 [24
]. The addition of ‘soft’, thiophilic metal ions (Cd2+
) capable of coordinating the phosphorothioate triggered 5′ SS cleavage, indicating that the isolated species were indeed B* complexes poised for first step catalysis. Although Cd2+
could rescue 5′ SS cleavage either in the presence or absence of ATP, Mg2+
only rescued splicing in the absence of ATP (albeit at a significantly reduced rate). While the slow reaction rate is consistent with Mg2+
being a poor phosphorothioate ligand, the ATP dependence suggests additional mechanisms at work. Koodathingal et al.
hypothesized that in the presence of ATP, the reaction was being proofread by a spliceosomal ATPase that shunted slow spliceosomes into a discard pathway prior to 5′ SS cleavage. In the absence of ATP this discard pathway was unavailable, so the slow spliceosomes could cleave the 5′ SS at their leisure. This purified system thus exhibits ‘kinetic proofreading’ [25
], in which the rate of a forward step in the spliceosome cycle is in competition with opening of a discard pathway by a spliceosomal ATPase ().
Figure 3 A model for the spliceosomal B* complex active site showing juxtaposition of several important groups during catalysis. In the work of Koodathingal et al., a site-specific phosphorthioate at position U80 in the U6 snRNA was used to stall spliceosomes (more ...)
Using yeast genetics, it had previously been shown that mutations in the DExD/H-box ATPase Prp16 can promote splicing of suboptimal substrates in vivo
, such as those containing branch point mutations [26
]. This made Prp16 an attractive target for proofreading 5′ SS cleavage, in addition to its known role in promoting the transition between C1 and C2 complexes [28
] (). Using the purified U6/U80-phosphorothioate spliceosomes and recombinant Prp16, Koodathingal et al.
were able to demonstrate both that Prp16 is directly responsible for this proofreading step and that the proofreading is reversible [24
]. If spliceosomes can enter and exit the Prp16 discard pathway multiple times, why aren’t all spliceosomes in the discard pathway eventually pulled into productive splicing reactions? The key appears to be irreversible destruction of spliceosomes in the discard pathway by an additional DExD/H-box ATPase, Prp43 ( and ). Using a pulse-chase approach and glycerol gradient centrifugation, Staley and coworkers have now shown that discarded spliceosomes at the stages of 5′ SS cleavage or exon ligation are substrates for Prp43 [24
]. Therefore, in addition to its roles in disassembling the I Complex () [30
] and in ribosome biogenesis [31
], Prp43 provides an irreversible step that prevents discarded spliceosomes from re-entering the splicing cycle.
In addition to reversible proofreading, we now know that the spliceosome can perform reverse splicing as well. While reverse splicing has long been known for self-splicing introns [33
], experimental evidence for reversible spliceosome chemistry had proven elusive until recently. By V5-epitope tagging a mutant of Prp22 defective in catalyzing spliced exon release, Tseng and Cheng were able to isolate in vitro
-assembled spliceosomes containing spliced exons and lariat intron product (spliced product complex, ) [34
]. By altering the KCl concentration in the absence of ATP, these spliceosomes could be coaxed into reversing both exon ligation and 5′ SS cleavage to re-generate pre-mRNA. Surprisingly, the monovalent ion requirements for the two reverse reactions were different – whereas KCl inhibited exon ligation reversal, it promoted 5′ SS cleavage reversal. Forward exon ligation was also favored by KCl meaning that spliceosomes in which exon ligation was first reversed by removing KCl could subsequently re-ligate the exons upon KCl addition. These results are consistent with the spliceosome being able to toggle between different catalytic conformations that favor forward or reverse progress through one or the other chemical reaction () [34