p68 RNA helicase is an essential human splicing factor that acts at the U1 snRNA-5' splice site duplex

Abstract

Modulation of the interaction between U1 snRNP and the 5' splice site (5'ss) is a key event that governs 5'ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5'ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5'ss during early stage of splicing. However, dissociation of the U1 from the 5'ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5'ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

FIG. 1.

(A) Immunoprecipitation of MB-cross-linked p65. The MB cross-linking mixture was electrophoresed by SDS-PAGE after no further treatment (lanes 1 and 2) or after immunoprecipitation with BSA (lane 3), monoclonal anti-β-galactosidase antibody (lane 4), or the antibody PAb204 (lane 5). (B) Western blot analyses of HeLa nuclear extracts (lane 1), mock-depleted (M) (protein G) HeLa nuclear extracts (lane 2), p68-depleted HeLa nuclear extracts (lane 3), and the p68-depleted HeLa extracts in which the His-tagged p68 RNA helicase is added (lane 4). The antibody PAb204 is used in the Western analyses. (C) MB cross-linking of recombinant p68 to the U1-5′ss duplex. MB cross-linking of p68 RNA helicase to RNA GC+DX/XhoI. The MB cross-linking reactions were carried out in mock-depleted (M) HeLa nuclear extracts (lane 1), p68-depleted HeLa nuclear extracts (lane 2), the p68-depleted HeLa extracts in which BSA was added to a concentration of 100 ng/μl (lane 3), or the p68-depleted HeLa extracts in which the His-tagged p68 was added to a concentration of 25 ng/μl (lane 4). Lane 5 is the precipitation of MB cross-linking by Ni-NTA bead. The cross-linking is carried out in the His-tagged p68-reconstituted HeLa nuclear extracts. IP, immunoprecipitation.

FIG. 2.

In vitro splicing of GC+DX transcripts in HeLa nuclear extracts. Splicing reactions were carried out under standard conditions. (A) Splicing extracts were untreated (lane 1), mock depleted (M) (lane 2), immunodepleted with anti-β-galacotosidase antibody (lane 3), or immunodepleted with PAb204 (lane 4). Lane M shows molecular weight markers. (B). Splicing extracts were mock depleted (M) (lane 2), immunodepleted with PAb204 (lane 3), immunodepleted with PAb204 where BSA was added to a concentration of 100 ng/μl (lane 4), immunodepleted with PAb204 where NS3 was added to 50 ng/μl (lane 5), or immunodepleted with PAb204 where His-tagged p68 was added to 25 ng/μl (lane 6). Lane 1 is the GC+DX transcript. The identities of the various RNA bands are shown schematically. IP, immunoprecipitation.

FIG. 3.

In vitro splicing of transcripts pPIP10A (A) and βG2 (B). Splicing reactions were carried out under standard conditions. Splicing extracts were mock depleted (M) (lane 2), immunodepleted with PAb204 (lane 3), immunodepleted with PAb204 where BSA was added to a concentration of 100 ng/μl (lane 4), immunodepleted with PAb204 where NS3 was added to 50 ng/μl (lane 5), or immunodepleted with PAb204 where His p68 was added to 25 ng/μl (lane 6). Lane 1 is the transcript pPIP10A (A) or βG2 (B), respectively.

FIG. 4.

(A) RNase H cleavage protection assays. Splicing reactions were performed with pPIP10A in 40% HeLa nuclear extracts (lanes 2 to 4) or extracts from which p68 RNA helicase was depleted (lanes 5 to 7). The extracts were pretreated with either RNase H and DNA oligonucleotide αU1_1-13 (lanes 3, 4, 6, and 7) or no RNase H treatment (lane 2 and 5). In the RNase H cleavage assays, the DNA oligonucleotide α5′ss was added (lanes 2, 3, 5, and 6), or random sequence DNA oligonucleotide Act1 was added (lanes 4 and 7). Lane 1 is the pre-mRNA pPIP10A without any treatment. (B) The U1 snRNA-pre-mRNA trioxsalen cross-linking. Trioxsalen cross-linking was performed with intact HeLa nuclear extracts (lanes 1 to 9) or with the extracts from which p68 RNA helicase was immunodepleted (lanes 10 to 14). The splicing was carried out for the indicated times before the trioxsalen was added to the splicing reactions and photolyzation under UV light. Lane 1 shows splicing alone without trioxsalen cross-linking. Lane 3 shows cross-linked RNAs that were further treated with RNase H in the presence of DNA oligonucleotide αU1 that is complementary to U1 64-75 (αU1_64-75). Lane 4 shows the cross-linked RNAs that were further treated with RNase H in the presence of random sequence DNA oligonucleotide Act1. Lane 15 is the U1 snRNA-pre-mRNA trioxsalen cross-linking in the extracts in which the endogenous p68 RNA helicase was replaced by 25 ng of recombinant p68 RNA helicase/μl.

FIG. 5.

Electrophoretic separation of spliceosome complexes. (A) Spliceosome complexes assembled in the immunodepleted HeLa nuclear extracts. Splicing reactions were carried out with the splicing substrate pPIP7A in the absence of ATP (lane 1). Splicing reactions were carried out in the extracts that were untreated (lane 2), mock depleted (lane 3), immunodepleted with anti-β-galactosidase antibody (lane 4), and immunodepleted with the antibody PAb204 (lane 5). All the splicing reactions were incubated at 30°C for 30 min. (B) Spliceosome complexes assembled in splicing time courses. Splicing reactions were carried out with splicing substrate pPIP10A in intact HeLa nuclear extracts (lanes 1 to 4) and the p68-depleted extracts (lanes 5 to 8) for the indicated times. Lane 9 presents the spliceosome complexes assembled in a splicing reaction with pPIP10A in the p68-depleted extracts in which the recombinant p68 was added to a concentration of 25 ng/μl. (C) Quantitative analyses of spliceosome complexes assembled on the splicing substrate pPIP10A. Line 1 (□) is the ratio of [H]/([H] + [A]) in the p68-depleted HeLa nuclear extracts. Line 2 (▴) is the ratio of [B/C]/([A] + [B/C]) in the intact HeLa extracts. Line 3 (X) is the ratio of [H]/([A] + [H]) in the intact HeLa extracts. Line 4 (▪) is the ratio of [H]/([H] + [A] + [B/C]) in the intact HeLa extracts. The quantity of each complex, [H], [A], and [B/C], was measured by volume integration of each complex band using a phosphorimager system.