We have used a new strategy to show that a ubiquitin mutant (I44A) with diminished capacity for protein-protein interactions strongly inhibits pre-mRNA splicing in vitro
. This inhibition can be rescued by removing the C-terminal tail of the I44A mutant ubiquitin, indicating that I44A ubiquitin inhibits splicing through its conjugation to a target protein. Further analysis of the splicing defect revealed that I44A ubiquitin diminishes steady-state levels of the U4/U6-U5 triple snRNP, which in turn stalls spliceosome assembly at the B complex (pre-spliceosome). Corroborating these results, ubistatin A, which binds to the I44-containing surface of ubiquitin33
, mimics the apparent dominant-negative effect of the mutant ubiquitin. Furthermore, the reduction in U4/U6-U5 triple snRNP levels in both cases can be attributed to an acceleration of U4/U6 unwinding that is likely to result from occlusion of conjugated ubiquitin. Moreover, pretreatment of purified U4/U6-U5 triple snRNPs with the deubiquitinating enzyme USP2 similarly accelerates U4/U6 unwinding, providing further evidence that ubiquitin represses U4/U6 unwinding directly. Indeed, a triple snRNP component (Prp8) that is already implicated in controlling U4/U6 unwinding3
is ubiquitinated in affinity-purified particles, suggesting a specific mechanism by which ubiquitin represses U4/U6 unwinding. Prp8-ubiquitin conjugates have also been identified, using a distinct methodology, in samples enriched for U4/U6-U5 triple snRNPs (S. Stevens, personal communication).
Our finding that USP2, I44A ubiquitin and ubistatin A each accelerate U4/U6 unwinding strongly suggests that ubiquitin normally serves to suppress U4/U6 unwinding by Brr2, thus stabilizing the triple snRNP under steady-state conditions (). We cannot exclude the possibility that ubiquitin recognition also has a role in promoting triple snRNP formation. Our detection of a Prp8-ubiquitin conjugate, and our previous demonstration of ubiquitin binding activity by Prp8’s Jab1/MPN domain16
, suggest a model in which Prp8 establishes an intramolecular interaction between its Jab1/MPN domain and conjugated ubiquitin. Prp8’s affinity for ubiquitin is decreased with the I44A mutant16
, and this could readily account for I44A ubiquitin’s ability to disrupt triple snRNP accumulation. Furthermore, mutations in Prp8 that compromise its ubiquitin binding activity also diminish U4/U6-U5 triple snRNP levels16
. In some cases, UBDs promote the ubiquitination of the proteins in which they reside, can limit the conjugation reaction to the addition of a single ubiquitin and can probably mediate intramolecular ubiquitin interactions39
; such roles are possible for the Jab1/MPN domain of Prp8.
Figure 7 A model for ubiquitin’s involvement in U4/U6-U5 triple snRNP accumulation and pre-mRNA splicing. The upper cycle depicts U4/U6-U5 triple snRNP assembly and disassembly23. The Brr2 ATPase promotes triple snRNP disassembly by catalyzing the unwinding (more ...)
We speculate that the formation of an intramolecular interaction between the Jab1/MPN domain of Prp8 and a conjugated ubiquitin could alter the conformation of Prp8 in a manner that diminishes its ability to activate U4/U6 unwinding by Brr2. Numerous subregions of Prp8 and Brr2 (and their human orthologs) have been shown to interact directly20
. The regions of Prp8 that interact with Brr2 include the C-terminal portion20
(which contains the ubiquitin-interacting Jab1/MPN domain16
) and also an N-terminal domain. Notably, these terminal regions of Prp8 also interact with each other20
. If the temporal modulation of these multiple interactions helps to specify the functional state of the spliceosome, as seems likely3
, then the formation and dissolution of ubiquitin–Jab1/MPN domain interactions could account for a subset of these structural and functional switches.
What catalyzes the ubiquitination events that regulate triple snRNP dynamics? Prp19 is unique in that it is both an essential splicing factor and an E3 ubiquitin ligase14
, although it is possible that other E3 splicing factors remain to be discovered. No in vivo
targets of Prp19 E3 ubiquitin ligase activity have been reported, but our evidence that Prp8-ubiquitin conjugates exist within purified triple snRNPs suggests that Prp8 could be a target of Prp19’s E3 ubiquitin ligase activity. Although our studies indicate that ubiquitin recognition helps to maintain U4/U6-U5 levels, previous work has shown that extracts immunodepleted of Prp19-containing complexes are competent for addition of the triple snRNP to the pre-spliceosome42
. However, proteins ubiquitinated by Prp19 before depletion could remain in the extract after depletion and could conceivably participate in the maintenance of the U4/U6-U5 triple snRNP. The existence of a postsplicing 35S complex in humans containing Prp19, other components of the Prp19-containing NineTeen Complex (NTC) and U5 snRNP proteins12
raises the possibility that Prp19-mediated ubiquitination of potential U5 snRNP protein targets could occur in this complex during spliceosome recycling, with the ubiquitinated form persisting through the reformation of the U4/U6-U5 triple snRNP.
It is possible that ubiquitin’s role in controlling triple snRNP disassembly is partially redundant with that of Snu114 and its GTPase cycle4
, given that both ubiquitin and GDP repress U4/U6 unwinding. Genetic interactions between snu114
), all of which have been linked to ubiquitin13
, could (together with our own work) reflect a network of physical and functional interactions that connect Prp19, ubiquitin, the U5 snRNP and splicing complex assembly and disassembly. Potential redundancy between the Prp19 ubiquitin conjugation cycle and the Snu114 GTPase cycle in controlling triple snRNP dynamics43
could help to rationalize several puzzling observations, including (i) the persistence of robust splicing in vitro
even after substantial ubiquitin depletion (), (ii) the apparent dispensability of Prp19 during early stages of spliceosome assembly42
and (iii) the inability of GTP and GDP to modulate in vitro
splicing activity in whole extracts, despite the well-established in vitro
role of the Snu114 GTPase4
Our discovery that ubiquitin recognition governs triple snRNP disassembly in no way precludes additional roles for ubiquitin in splicing. Several lines of evidence indicate that U4/U6 unwinding in the triple snRNP is mechanistically related to U4/U6 unwinding during spliceosome activation and to U2/U6 unwinding during spliceosome disassembly: all of these events seem to be driven by the Brr2 ATPase4
, and all are functionally linked to Snu114 and its guanine-nucleotide binding state4
. It is therefore possible that the role of ubiquitin recognition in inhibiting triple snRNP disassembly is recapitulated later in the splicing cycle: ubiquitin-UBD interactions may continue to repress U4/U6 unwinding during spliceosome assembly until the U4/U6 unwinding stage during spliceosome activation, or it may prevent the premature unwinding of U2/U6 during the catalytic steps until spliceosome disassembly, or both (). A possible role in repressing premature U2/U6 unwinding is especially attractive, given the observation that Prp19 depletion leads to the premature departure of U5 and U6 snRNAs from the spliceosome following U4/U6 unwinding and catalytic activation42
. Furthermore, ubistatin A promotes the disassembly of the U2/U6-U5–intron postsplicing complex4
(E.C.S. and J.P.S., unpublished data), and apparent ubiquitin conjugates in post-splicing complexes have been detected in budding yeast (S. Stevens, personal communication). The activities of E3 ubiquitin ligases such as Prp19 are frequently counteracted by deubiquitinating enzymes9
, and this may represent a mechanism for derepressing Brr2-dependent spliceosome dynamics.
Dynamic, multicomponent complexes that function in gene expression generally adopt several mechanisms to ensure efficiency and accuracy. The spliceosome is no exception. Our observation that ubiquitin recognition is important for maintaining U4/U6-U5 triple snRNP levels uncovers a discrete stage of spliceosome assembly that is modulated by ubiquitin. This not only points to a previously unrecognized means of regulating the splicing machinery, but also expands the scope of ubiquitin regulation into a new biochemical context.