The RNA helicase DDX39 contributes to the nuclear export of spliceosomal U snRNA by loading of PHAX onto RNA

Abstract

RNA helicases are involved in RNA metabolism in an ATP-dependent manner. Although many RNA helicases unwind the RNA structure and/or remove proteins from the RNA, some can load their interacting proteins onto RNAs. Here, we developed an in vitro strategy to identify the ATP-dependent factors involved in spliceosomal uridine-rich small nuclear RNA (U snRNA) export. We identified the RNA helicase UAP56/DDX39B, a component of the mRNA export complex named the transcription-export (TREX) complex, and its closely related RNA helicase URH49/DDX39A as the factors that stimulated RNA binding of PHAX, an adapter protein for U snRNA export. ALYREF, another TREX component, acted as a bridge between PHAX and UAP56/DDX39B. We also showed that UAP56/DDX39B and ALYREF participate in U snRNA export through a mechanism distinct from that of mRNA export. This study describes a novel aspect of the TREX components for U snRNP biogenesis and highlights the loading activity of RNA helicases.

Graphical Abstract

Figure 1.

ATP is required for efficient RNA binding of PHAX in HeLa nuclear extracts (A) Schematic diagram of the in vitro PHAX-RNA binding assay. (B) A mixture of in vitro-transcribed ³²P-labeled RNAs containing fushitarazu (ftz) mRNA, U1ΔSm, U5ΔSm and U6Δss small nuclear RNAs (snRNAs) was incubated with HeLa nuclear extracts (HNEs) in the presence or absence of ATP, then RNA co-immunoprecipitation (co-IP) assays were performed using an anti-PHAX or anti-mouse IgG antibody. U6Δss snRNA was uncapped, while the other RNAs were m⁷G-capped. Precipitated RNAs were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. (C) The quantification of relative IP efficiency from three independent experiments is shown. IP efficiencies were determined by dividing the amount of immunoprecipitated RNA by the amount of each RNA input. Averages and standard deviations are noted. IP efficiency in the presence of ATP was set to 1. P-values were calculated by a two-tailed t-test. (D) Precipitated proteins were analyzed by SDS-PAGE and western blotting. ³²P-labeled RNAs were not included in this assay. TBP: TATA-binding protein. (E) The quantification of IP efficiency from three independent experiments is shown. IP efficiencies were determined by dividing the amount of immunoprecipitated protein by the amount of each protein input. Averages and standard deviations are noted. nd: not detected.

Figure 2.

Identification of candidate helicases for ATP-dependent loading of PHAX (A) Schematic diagram of the process for identifying candidate helicases. HNEs were incubated with GST-PHAX, His-CBP80 and His-CBP20 in the presence of ATP and RNase A, followed by GST pull-down and HRV 3C protease treatment. PHAX-interacting proteins were irradiated by ultraviolet (UV) light and detected by SDS-PAGE and autoradiography. (B) The two bands corresponding to 55 kDa and 100 kDa were named p55 and p100, respectively. (C) The molecular weight (Mw) and theoretical isoelectric point (pI) of RNA helicases of ∼55 kDa are shown. Mw and theoretical pI were obtained from the ExPASy Compute pI/Mw tool. (D) The irradiated sample in (B) was immunoprecipitated using antibodies against UAP56 and DBP5, then detected by SDS-PAGE and autoradiography. (E) HNEs were subjected to GST pull-down assay using GST or GST-PHAX in the presence of RNase A followed by western blotting.

Figure 3.

ATP-bound UAP56 stimulates RNA binding of PHAX (A) Schematic diagram of the in vitro PHAX-RNA binding assay. (B) A mixture of in vitro-transcribed ³²P-labeled RNAs containing dihydrofolate reductase (DHFR) mRNA, U1ΔSm small nuclear RNA (snRNA), U5ΔSm snRNA, U6Δss snRNA, and tRNA^Phe was incubated with HeLa nuclear extracts (HNEs), ATP, either GST or GST-PHAX, and either buffer, FLAG-UAP56 WT, or K95E mutant. Then, GST pull-down assays were performed. U6Δss snRNA and tRNA^Phe were uncapped, while the other RNAs were m⁷G-capped. Pulled down RNAs were analyzed by denaturing PAGE and autoradiography. (C) The quantification of pull-down efficiency of DHFR mRNA, U1ΔSm snRNA, U5ΔSm snRNA and tRNA^Phe from four independent experiments performed as in (B). Pull-down efficiencies were determined by dividing the amount of pulled down RNAs by the amount of each RNA input. Averages and standard deviations are noted. P-values were calculated by a two-tailed t-test. (D) Pulled down proteins were analyzed by SDS-PAGE and western blotting. ³²P-labeled RNAs were not included in this assay. TBP: TATA-binding protein. (E) A mixture of in vitro-transcribed ³²P-labeled RNAs containing U1ΔSm, U5ΔSm, and U6Δss snRNAs was incubated with HNEs, ATP, GST-PHAX and either buffer, FLAG-UAP56 (39B), FLAG-URH49 (39A), or FLAG-DBP5 (19). Then, GST pull-down assays were performed. Pulled down RNAs were analyzed by denaturing PAGE and autoradiography. Pulled down proteins were analyzed by SDS-PAGE and western blotting. ³²P-labeled RNAs were not included in this protein–protein binding assay. (F) Quantification of pull-down efficiency of U1ΔSm, U5ΔSm and U6Δss snRNAs from three independent experiments performed as in (E). Pull-down efficiencies were determined by dividing the amount of pulled down RNAs by the amount of each RNA input. Averages and standard deviations are noted. P-values were calculated by a two-tailed t-test. *P< 0.05, ns: not significant.

Figure 4.

UAP56 interacts with PHAX in an ATP-dependent manner (A) HeLa nuclear extracts (HNEs) and RNase A were incubated with GST or GST-PHAX in the presence or absence of ATP. Pulled down proteins were detected by western blotting using anti-UAP56 and anti-CBP80 antibodies. The gel after blotting was stained with Coomassie brilliant blue (CBB). (B) HNEs, GST-PHAX, and RNase A were incubated with FLAG-UAP56 WT or K95E mutant in the presence or absence of ATP. Pulled down proteins were detected by western blotting using an anti-FLAG antibody. The gel after blotting was stained with CBB. (C) HNEs and RNase A were incubated with either GST or GST-PHAX and either FLAG-UAP56 WT or K95E mutant in the presence or absence of ATP. Pulled down proteins were detected by western blotting using an anti-FLAG antibody. The gel after blotting was stained with CBB. Uncropped CBB staining is shown in Supplementary Figure S6.

Figure 5.

Identification of bridging factors between PHAX and UAP56 (A) Schematic diagram of the process for identifying bridging factors. (B) HeLa nuclear extracts (HNEs) and RNase A were incubated with either buffer or GST-PHAX and either buffer or FLAG-UAP56, in the presence of ATP. Immunoprecipitation (IP) experiments were then performed. The bound proteins were eluted with a FLAG peptide. The eluate was subjected to GST pull-down. The bound proteins were treated with HRV 3C protease. The supernatant was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry or western blotting using an anti-ALY antibody. (C) Identified proteins specific to lane 4 of (B) are listed. Components of the TREX complex are shown in red text.

Figure 6.

ALY bridges between PHAX and ATP-bound UAP56 (A) HNEs and RNase A were subjected to protein A Sepharose that was pre-bound to rabbit antibody against mouse IgG (control) or ALY (ΔALY). The depleted HNEs were incubated with GST-PHAX in the presence or absence of ATP, and GST pull-down was performed. The bound proteins were analyzed by SDS-PAGE and western blotting using antibodies against UAP56, ALY or CBP80. The gel after blotting was stained with CBB. (B) Purified recombinant GST or GST-PHAX was incubated with FLAG-UAP56 and RNase A in the presence or absence of FLAG-ALY and ATP, and GST pull-down was performed. The pulled down proteins were analyzed by SDS-PAGE and western blotting using an anti-FLAG antibody. The relative pull-down efficiency of FLAG-UAP56 and FLAG-ALY is shown. The gel after blotting was stained with CBB. (C) Purified recombinant GST or GST-ALY was incubated with T7-PHAX and RNase A, and GST pull-down was performed. Pulled down proteins were analyzed by SDS-PAGE and western blotting using an anti-T7 antibody. The gel after blotting was stained with CBB. (D) Schematic representation of the interaction between PHAX and UAP56 via ALY. Uncropped CBB staining is shown in Supplementary Figure S7.

Figure 7.

UAP56 and ALY are involved in U small nuclear RNA (snRNA) export (A) Schematic diagram of Xenopus oocyte microinjection. (B) The same RNA mixture as in Figure 3 was injected with a rabbit antibody against mouse IgG (control), UAP56, or ALY into the nucleus. U6Δss snRNA and tRNA^Phe were uncapped, while the other RNAs were m⁷G-capped. U6Δss does not leave the nucleus and served as an internal control for nuclear integrity. RNA was immediately extracted from nuclear (N) and cytoplasmic (C) fractions (lanes 1 and 2) or 3.5 h (lanes 3–8) after the injection, and then analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. (C) Quantification of the export of dihydrofolate reductase mRNA (DHFR), U1ΔSm snRNA (U1), U5ΔSm snRNA (U5), and tRNA^Phe from three independent experiments performed in (B). The export efficiency of the control was set to 100%. Averages and standard deviations are noted. P-values were calculated by comparisons with the control: *P< 0.05, ***P< 0.001, ns: not significant. (D) The same RNA mixture was injected with BSA-NES (NES) or BSA-NES M10 mutant (NES mut). BSA-NES, but not NES M10 mutant, saturates CRM1 export (61). RNA export was analyzed as in (B). (E) Quantification of the export of DHFR mRNA, U1ΔSm snRNA, U5ΔSm snRNA, and tRNA^Phe from three independent experiments performed in (D). The export efficiency of the control was set to 100%. Averages and standard deviations are noted. P-values were calculated by comparisons with the NES mut: ***P< 0.001, ns: not significant. (F) The same RNA mixture was injected with CTE RNA (CTE) or CTE M36 mutant RNA (CTE mut). CTE, but not CTE M36 mutant, binds specifically to TAP and inhibits the TAP-dependent export (62). RNA export was analyzed as in (B). (G) Quantification of the export of DHFR mRNA, U1ΔSm snRNA, U5ΔSm snRNA, and tRNA^Phe from three independent experiments performed in (F). Averages and standard deviations are noted. P-values were calculated by comparisons with the CTE mut: ***P< 0.001, ns: not significant.

Figure 8.

RNA binding and ATPase activities of UAP56 (A) In vitro-transcribed ³²P-labeled U1ΔSm and U5ΔSm snRNAs were incubated with or without purified recombinant FLAG-UAP56 (1 μM) in the absence or presence of 2 mM nucleotide (ATP, ADP or ATP-γS) and 2 mM MgCl₂, then RNA co-immunoprecipitation (co-IP) assays were performed using an anti-FLAG M2 antibody. Precipitated RNAs were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. The quantification of IP efficiency from three independent experiments is shown. IP efficiencies were determined by dividing the amount of immunoprecipitated RNA by the amount of each RNA input. Averages and standard deviations are noted. (B) [α-³²P] ATP was incubated with or without purified recombinant FLAG-UAP56 in the absence or presence of U5ΔSm RNA at 37°C. Products were developed by thin layer chromatography. ATP and ADP were detected by autoradiography. Quantification of ATP hydrolysis efficiency from three independent results is shown. Averages and standard deviations are noted. Relative ATPase efficiency without U5 RNA was set to 1. (C) A model of the mechanism by which UAP56 loads PHAX onto U small nuclear RNA (snRNA). In mRNA export, UAP56 loads the TREX complex onto mRNA and then recruits TAP-p15. UAP56 may dissociate from mRNA during TAP-p15 recruitment, leading to RNA export to the cytoplasm. Note that for the sake of simplicity, this model does not show the EJC and SR proteins as adaptors to recruit TAP-p15. In U snRNA, PHAX forms the U snRNA-type TREX complex (uTREX) with UAP56 and ALY and is loaded onto U snRNA as the uTREX complex. During the loading process, UAP56 may unwind the stable cap-proximal structure of U snRNA using energy from ATP hydrolysis. Immediately after loading and ATP hydrolysis, the uTREX complex components, except PHAX, may dissociate from RNA. The PHAX that remains then recruits CRM1-RanGTP, leading to RNA export. After dissociation, UAP56 is loaded with ATP again for the next round of loading of PHAX onto RNA.