ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome

Abstract

We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5' splice site (5'ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5'ss, while the mutants still interacted with the U1-5'ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5'ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.

FIG. 1.

(A) Schematic illustration of the sequence motifs in the helicase core of DEAD box RNA helicase and the p68 LGLD and HLIGR mutants. (B) Coomassie blue staining of SDS-PAGE of recombinant p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) that were expressed and purified from bacterial E. coli. (C) Cross-links of p68 wt (lane 1), LGLD mutant (lane 2), or HILGR mutant (lane 3) to [α-³²P]ATP were analyzed by SDS-PAGE followed by autoradiography. (D) RNA unwinding by p68 wt (lane 3), LGLD mutant (lane 4), or HILGR mutant (lane 5) was analyzed by SDS-PAGE followed by autoradiography. Lane 1 is duplex RNA denatured by heating to 95°C. Lane 2 is the duplex RNA. (E) ATPase activities of p68 wt/mutant (indicated) were measured by colorimetric assay. The ATPase activity (y axis) is expressed as μM P_i/mg of p68 or mutants.

FIG. 2.

Splicing of transcript pPIP10A in HeLa nuclear extracts in which the endogenous p68 is mock depleted (lane 2) or depleted by antibody against p68 (lanes 3 to 5). The dephosphorylated recombinant p68 (Dp), protein buffer (lane 2), wild type (lane 3), mutant HLIGR (lane 4), or mutant LGLD (lane 5) was added to the p68-depleted extracts. Lane 1 is the pre-mRNA (pPIP10A) without splicing.

FIG. 3.

Trioxsalen cross-linking of the U1 snRNA to pre-mRNA in HeLa nuclear extracts. The U1-pre-mRNA cross-linking band is indicated. The cross-linking reactions were carried out in the intact extracts (lanes 2 to 5), p68-depleted extracts (lanes 6 to 20), and p68-depleted extracts to which wild-type p68 (lanes 11 to 15), mutant HLIGR (lanes 6 to 10), or mutant LGLD (lanes 16 to 20) was added. The splicings were carried out for the indicated times before the trioxsalen was added to the splicing reactions. Lane 4 is cross-linked RNAs that are further treated with RNase H in the presence of DNA oligonucleotide αU1 that is complementary to U1 64-75 (αU1_64-75). Lane 5 is the cross-linked RNAs that are further treated with RNase H in the presence of random sequence DNA oligonucleotide Act1.

FIG. 4.

(A) MB cross-linking of wild-type (left panel), mutant LGLD (middle panel), or mutant HLIGR (right panel) His-p68 to the radiolabeled transcript pGC+DX in endogenous p68-depleted HeLa nuclear extracts. The splicings are carried out for the indicated times before the methylene blue is added to the splicing reactions. The lane marked p68 is the MB cross-linking carried out in intact HeLa nuclear extracts without addition of recombinant p68. (B) The amount of p68 in each cross-linking reaction was determined by immunoblotting of the same MB cross-link SDS-PAGE with anti-His antibody.

FIG. 5.

(A) Electrophoretic separation of spliceosome complexes. Splicing reactions were carried out with splicing substrate pPIP10A in the extracts: untreated (lane 1), p68 was depleted with antibody against p68 (lane 2), p68 was depleted and recombinant wt p68 was added (lane 3), p68 was depleted and recombinant LGLD mutant was added (lane 4), and p68 was depleted and recombinant HLIGR mutant was added (lane 5). All of the splicing reactions were incubated at 30°C for 30 min. (B) Assembly of wt p68 (lane 2), the LGLD mutant (lane 3), or the HLIGR mutant (lane 4) into spliceosome complexes in p68-depleted extracts was detected by immunoblotting of recombinant p68 using antibody against His₆ tag. The recombinant p68s are dephosphorylated before adding to the splicing reactions. Lane 1 is the complexes assembled in the extracts without addition of recombinant p68. (C) Spliceosome complex assembly in p68-depleted HeLa nuclear extracts to which wt p68 was added (lanes 2 to 4 and 11 and 12), mutant LGLD was added (lanes 5 to 7 and 13 and 14), or mutant HLIGR was added (lanes 8 to 10 and 15 and 16). The splicing reactions were carried out for the time indicated.

FIG. 6.

Functional role of p68 in the pre-mRNA splicing process in vivo. (A) Knockdown of p68 by RNA interference and exogenous expression of HA-p68 wt/mutant in HT-29 cells were analyzed by immunoblotting using anti-p68 antibody (upper panel) or anti-HA antibody (bottom panel). NT means the cells were treated with nonspecific siRNA duplex (control). (B) Double reporter assays for the ratio of spliced/total pre-mRNA in HT-29 cells in which p68 was knocked down and wt or mutant p68 (indicated) was expressed. The spliced/total pre-mRNA ratio is expressed as the luciferase activity divided by β-galactosidase activity (31). (C and D) RT-PCR probe of the spliced/unspliced mRNA of β-actin and GAPDH genes in HT-29 cells in which p68 was knocked down and wt or mutant p68 (indicated) was expressed. A pair of primers spanning exon 4 and exon 5 (C) or exon 4 and intron 4 (D) was used in the RT-PCRs.

FIG. 7.

RNA-IP of spliced/unspliced β-actin and GAPDH mRNA in HT-29 cells in which wt or mutant HA-p68 (indicated) is expressed. RNAs were precipitated by anti-HA antibody. (A) Expression of HA-p68 wt/mutant was detected by immunoblotting with anti-HA antibody. (C) The immunoprecipitated RNAs were detected by RT-PCR using a pair of primers crossing exon 4 and intron 4 of β-actin (upper panel) or GAPDH (bottom panel) genes. (D) The immunoprecipitated RNAs were detected by RT-PCR using primers crossing both exon 4 and intron 4 or exon 4 and exon 5 (indicated) of β-actin (upper panel) or GAPDH (bottom panel) genes. (E) The immunoprecipitated RNAs were detected by RT-PCR using a pair of primers annealed to histone 2B or CEBP (Table 1) genes. IN indicates input, where the RT-PCRs were performed with total RNA extracts without IP. IP indicates that the RT-PCRs were performed with the RNAs that were coprecipitated with anti-HA antibody. RN indicates the immunoprecipitated mixtures were treated with RNase before performing RT-PCRs. (B) The immunoprecipitated RNAs were detected by RT-PCR or PCR using the same primers as in panel C or D.