A role for ubiquitin in the spliceosome assembly pathway

Abstract

The spliceosome uses numerous strategies to regulate its function in mRNA maturation. Ubiquitin regulates many cellular processes, but its potential roles during splicing are unknown. We have developed a new strategy that reveals a direct role for ubiquitin in the dynamics of splicing complexes. A ubiquitin mutant (I44A) that can enter the conjugation pathway but is compromised in downstream functions diminishes splicing activity by reducing the levels of the U4/U6-U5 small nuclear ribonucleoprotein (snRNP). Similarly, an inhibitor of ubiquitin's protein-protein interactions, ubistatin A, reduces U4/U6-U5 triple snRNP levels in vitro. When ubiquitin interactions are blocked, ATP-dependent disassembly of purified U4/U6-U5 particles is accelerated, indicating a direct role for ubiquitin in repressing U4/U6 unwinding. Finally, we show that the conserved splicing factor Prp8 is ubiquitinated within purified triple snRNPs. These results reveal a previously unknown ubiquitin-dependent mechanism for controlling the pre-mRNA splicing pathway.

Figure 1

A mutant form of ubiquitin (Ub) inhibits pre-mRNA splicing activity in vitro. (a) Splicing extract from a Saccharomyces cerevisiae strain expressing His₆-myc–tagged ubiquitin was depleted of ubiquitin by Ni²⁺ affinity chromatography. Ubiquitin levels before and after depletion were assayed by probing a western blot with anti-myc antibodies. Hexokinase (below) was used as a control. (b) Ubiquitin-depleted splicing extract was used in 30-min in vitro splicing assays following the addition of wild-type and mutant forms of ubiquitin, as specified above each lane. Each ‘ΔCtail’ mutant had a four-amino-acid truncation at its C terminus to prevent entry into the target conjugation pathway. The mobilities of pre-mRNA, splicing intermediates and splicing products are indicated on the right. (c) Data from b and two equivalent splicing experiments were quantitatively analyzed with a PhosphorImager, and splicing efficiency (defined as (spliced products + splicing intermediates)/total radiolabeled RNA, normalized to the nonsupplemented extract) was plotted. The identities of the added ubiquitin derivatives are given at the bottom. Error bars indicate the range of the three experiments.

Figure 2

The U2 snRNP-containing pre-spliceosome accumulates in the presence of I44A ubiquitin. (a) Radiolabeled pre-mRNA was incubated in splicing extract for 30 min, and the assembled complexes were then analyzed by native gel electrophoresis. The mobilities of H (nonspecific), B (U2), A2 (U2-U4/U6-U5 and U2/U6-U5), and A1 (U2/U6-U5) complexes are indicated on the right. The kinetically distinct A2-1 and A2-2 complexes were not resolved in this experiment. Lanes 1–4 were from control reactions that served to guide the identification of the complexes (lane 1, standard splicing reaction; lane 2, extract depleted of U2 snRNA by oligonucleotide-directed RNase H cleavage; lane 3, extract depleted of ATP by incubation with glucose; lane 4, extract supplemented with 5 mM EDTA, which leads to A1 complex accumulation47). Lanes 5–7 are from reactions with ubiquitin-depleted extract supplemented with buffer (lane 5), 1 mM wild-type ubiquitin (lane 6) or 1 mM I44A ubiquitin (lane 7). (b) As in a, except that the complete, U2 knockout, –ATP and EDTA reactions were omitted, and reactions supplemented with I44A ΔCtail ubiquitin (lane 4) and ΔCtail ubiquitin (lane 5) were included.

Figure 3

I44A ubiquitin blocks spliceosome assembly by interfering with U4/U6-U5 triple snRNP accumulation. Splicing extract from a strain expressing a triple-HA–tagged U5 snRNP component (Prp8^3HA) was immunoprecipitated (IP) with anti-HA antibodies following the addition of buffer (lane 3), 1 mM wild-type ubiquitin (lane 4) or 1 mM I44A ubiquitin (lane 5). Parallel control immunoprecipitations from an isogenic untagged strain are shown in lanes 6–8. RNAs extracted from the immunoprecipitates were used as templates in reverse-transcriptase reactions with radiolabeled primers specific to the U1, U2, U4, U5 and U6 snRNAs. The mobilities of the primer extension products are indicated on the right. Lane 1, radiolabeled DNA size markers; lane 2, primer extension with total RNA from the splicing extract used in lanes 3–5.

Figure 4

The ubiquitin (Ub) binding small molecule ubistatin A recapitulates the inhibitory effects of I44A ubiquitin. (a) Splicing extract was used in 30-min in vitro splicing assays following the addition of I44A ubiquitin (lane 4), 20 µM ubistatin A in DMSO (lanes 5–7) or DMSO alone (lanes 3, 8 and 9). In lanes 6–9, the ubistatin A or DMSO was preincubated with monoubiquitin or K48-linked tetraubiquitin chains before addition to the splicing extract. The mobilities of pre-mRNA, splicing intermediates and splicing products are indicated on the right. (b) Splicing extract from a strain expressing a triple-HA–tagged U5 snRNP component (Prp8^3HA) was immunoprecipitated (IP) with anti-HA antibodies following the addition of buffer (lanes 3 and 8), 1 mM wild-type ubiquitin (lanes 4 and 9), 1 mM I44A ubiquitin (lanes 5 and 10), DMSO (lane 13), 50 µM ubistatin A in DMSO (lanes 6 and 11) or 100 µM ubistatin A in DMSO (lanes 7 and 12). A parallel control immunoprecipitation from an isogenic untagged strain is shown in lane 14. In lanes 8–12, ATP was depleted after an initial 10-min incubation in the presence of endogenous ATP to permit ubiquitin activation. Extracted RNAs were used as templates in reverse-transcriptase reactions with radiolabeled primers specific to the U1, U2, U4, U5 and U6 snRNAs. The mobilities of the primer extension products are indicated on the right.

Figure 5

Conjugated mutant I44A ubiquitin or the deubiquitinating cysteine protease USP2 derepresses U4/U6 unwinding in purified U4/U6-U5 snRNPs. (a) Extract from a strain expressing TAP-tagged Brr2 (yJPS776) was incubated with wild-type or I44A ubiquitin in the presence of ATP to allow conjugation; glucose was then added to deplete ATP, and the U4/U6-U5 particle was immunopurified and assayed for U4/U6 unwinding. Cold-phenol–extracted RNA was fractionated by native gel electrophoresis and the U4 and U6 snRNAs were detected by northern blotting as described. The mobilities of annealed U4/U6 (corresponding to the triple snRNP) and free U4 and U6 (corresponding to the free U4 and U6 snRNPs) are indicated on the left. Reactions were incubated with ATP; with or without GDP; and with or without wild-type, tailless wild-type (Ubiquitin ΔCtail), mutant (I44A) or tailless mutant (I44A ΔCtail) ubiquitin, as indicated. (b) Data from a and a replicate experiment were quantified and plotted as shown. Error bars represent the range of the two experiments. (c) U4/U6-U5 particles were immunopurified from a Prp28-TAP strain (yJPS1004). The triple snRNP was preincubated with 2.5 µM of the ubiquitin deconjugating enzyme USP2 and in the indicated reaction USP2 itself was preincubated with 2.5 µM ubiquitin aldehyde, a USP2 inhibitor. All reactions were initiated with ATP, and GDP was included or omitted as indicated. U4/U6 unwinding was assayed as described. (d) Data from c and a replicate experiment were quantitated and plotted as shown. Error bars represent the range of the two experiments.

Figure 6

Affinity-purified U4/U6-U5 triple snRNPs contain Prp8-ubiquitin conjugates. U4/U6-U5 triple snRNP particles were immunopurified from a Brr2-TAP strain expressing wild-type ubiquitin (Ub, yJPS1274) or His₆-myctagged ubiquitin (His₆-myc-Ub, yJPS1275). Following TEV elution, the purified snRNPs were bound to Ni²⁺-NTA under denaturing conditions. The Ni²⁺-NTA–bound material (Bound), as well as one-tenth of the TEV eluates that were used in the Ni²⁺-NTA purification (10% input), were separated on a 4–20% protein gel and subjected to western analysis using antibodies against Prp8. Mobilities of molecular weight markers are given on the left.

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 disassembly. The Brr2 ATPase promotes triple snRNP disassembly by catalyzing the unwinding of the U4 and U6 snRNAs,,,. Our results indicate that the recognition of a Prp8-ubiquitin conjugate within the U4/U6-U5 triple snRNP suppresses Brr2-catalyzed disassembly. The lower cycle depicts a hypothetical model for ubiquitin’s involvement in the complete pre-mRNA splicing pathway,,. The U4/U6-U5 triple snRNP associates with the U2 snRNP-containing pre-spliceosome, and Brr2 can block this step by catalyzing triple snRNP disassembly; our results indicate that the recognition of a Prp8-ubiquitin conjugate suppresses Brr2 activity at this stage, allowing the triple snRNP to stably engage the pre-spliceosome. Once the spliceosome is assembled, the suppression of Brr2 activity is relieved (perhaps in part by the disruption of ubiquitin recognition), leading to U4/U6 unwinding and catalytic activation of the spliceosome. Upon spliceosome activation, Brr2 activity must once again be suppressed, to inhibit premature spliceosome disassembly probably due to Brr2-catalyzed U2/U6 unwinding. We speculate that ubiquitin recognition is again involved in Brr2 inhibition at this stage. Once the splicing reaction is complete, the suppression of Brr2 activity is again overcome (perhaps in part by the disruption of ubiquitin recognition), leading to U2/U6 unwinding and spliceosome disassembly (ref. ; E.C.S. and J.P.S., unpublished data). The points at which ubiquitin enters and exits the triple snRNP and spliceosome cycles are not known and are therefore omitted from the figure for clarity.