Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome

Abstract

The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56's ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF(65) and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56's dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF(65). Our results indicate that hUAP56 first interacts with U2AF(65) in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.

Figure 1.

Development of a biochemical complementation system to analyze the role of hUAP56 in spliceosome assembly. (A) Immunoblot analysis showing protein levels of hUAP56 and, as a specificity control, U2AF⁶⁵, in mock-depleted HeLa NE and in hUAP56-depleted HeLa NE (ΔUAP56NE). (B) hUAP56 was analyzed for its ability to complement splicing of the Ad ML pre-mRNA substrate in the ΔUAP56NE. Splicing of Ad ML in mock-depleted HeLa NE is shown as a control. The signal corresponding to fully spliced pre-mRNA was quantitated; for NE, the value was arbitrarily set to 100%, and for ΔUAP56NE and ΔUAP56NE+hUAP56 measured 2% and 64%, respectively. (C) Splicing of the β-globin pre-mRNA substrate following addition of hUAP56 to the ΔUAP56NE. Quantitation of the fully spliced pre-mRNA signal was as follows: NE, 100%; ΔUAP56NE, 3%; ΔUAP56NE+hUAP56, 63%.

Figure 2.

Requirement of the hUAP56 ATP-binding, ATPase, and dsRNA unwindase/helicase domains for in vitro pre-mRNA splicing. (A) Gel filtration profiles of wild-type hUAP56 or the ATP-binding (K95A), ATPase (E197A), or dsRNA unwindase/helicase (D199A) mutants. (mAU) milliabsorption unit. (B) Thermal melting curves for wild-type hUAP56 and mutant derivatives, as monitored by circular dichroism spectroscopy. The melting temperatures (T_m) for wild-type, K95A, E197A, and D199A hUAP56 are 55°C, 56°C, 52°C, and 53°C, respectively. (C) Wild-type hUAP56 or mutant derivatives were analyzed for their ability to support splicing of the Ad ML pre-mRNA substrate in the ΔUAP56NE.

Figure 3.

hUAP56 functions during both prespliceosome and spliceosome assembly. (A) Wild-type hUAP56 and mutant derivatives were analyzed for their ability to support assembly of the prespliceosome (complex A) and mature spliceosome (complexes B and C) in the ΔUAP56NE. Also shown is assembly of the nonspecific complex H. (B) The D199A mutant was analyzed for its ability rescue complex A formation in the presence and absence of U2 snRNA and ATP. (C) Mutant hUAP56 derivatives were analyzed for their ability to support complex E assembly. U2AF-depleted and mock-depleted nuclear extracts were analyzed as negative and positive controls, respectively.

Figure 4.

Role of hUAP56 in prespliceosome assembly. (A) hUAP56 recruitment experiments. Wild-type hUAP56 or mutant derivatives were added to the ΔUAP56NE, followed by addition of a biotinylated Ad ML pre-mRNA substrate. Splicing complexes were affinity-purified and analyzed by immunoblotting for hUAP56. (B) GST pull-down experiments. hUAP56 or mutant derivatives were incubated with GST-U2AF⁶⁵ and, following GST pull-down, bound hUAP56 was detected by immunoblotting. Protein–protein interaction experiments were performed in the presence or absence of ATP, as indicated. (C) GST pull-down experiments were performed as described in B, except that ATP was replaced by either the non-hydrolyzable ATP analog ATP-γS, ADP, or GTP.

Figure 5.

hUAP56 contacts U4 and U6 snRNAs. U snRNA cross-linking/immunoprecipitation experiments. (A, left) Primer-extension analysis in untreated HeLa NE shows the positions of U snRNAs. (Right) Following UV irradiation, hUAP56 was immunoprecipitated, and snRNAs in the immunoprecipitate were purified and detected by primer-extension analysis. (B) Wild-type hUAP56 or mutant derivatives were added to the ΔUAP56NE, and association with U4 and U6 snRNAs was analyzed by UV cross-linking/immunoprecipitation. (C) Recombinant hUAP56, U2AF⁶⁵, or both hUAP56 and U2AF⁶⁵ were added to HeLa NEs that had been depleted of either hUAP56, U2AF, or both hUAP56 and U2AF, and association of hUAP56 with U4 and U6 snRNAs was analyzed by UV cross-linking/immunoprecipitation. (D) The ability of hUAP56 to bind U4 and U6 shRNAs was tested in the presence and absence of an unlabeled Ad ML pre-mRNA.

Figure 6.

hUAP56 can unwind the U4/U6 duplex. (A) Native gel assay. Total RNA was purified from extracts, incubated in the presence or absence of Ad ML pre-mRNA, and fractionated on a native gel to separate free U4 and U6 snRNAs from the U4/U6 duplex. Gels were probed with either a U4 (top) or U6 (bottom) probe. The ratio of U4/U6 duplex to free U4 or U6 was quantitated and is shown. (B) Psoralen cross-linking analysis. Assays were performed in the ΔUAP56NE following addition of wild-type hUAP56 or mutant derivates. (C) hUAP56 was depleted from U2AF-depleted extracts, and psoralen cross-linking experiments were performed following addition of hUAP56, U2AF⁶⁵, or a combination of hUAP56 and U2AF⁶⁵. (D) Schematic diagram of the hUAP56 protein, showing the residues required for ATP-binding, ATPase, and dsRNA unwindase/helicase activities and their role in prespliceosome and mature spliceosome assembly.