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Sip1, a novel RS domain-containing protein essential for pre-mRNA splicing - PubMed

Sip1, a novel RS domain-containing protein essential for pre-mRNA splicing

W J Zhang et al. Mol Cell Biol. 1998 Feb.

Abstract

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5' and 3' splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.

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Figures

FIG. 1
FIG. 1

Amino acid sequence of Sip1 predicted from the full-length cDNA sequence. SR or RS dipeptides are underlined. Eight imperfect repeats of RRSRSXSX are in boldface and underlined. The CTD-binding motif is doubly underlined.

FIG. 2
FIG. 2

Northern analysis of Sip1 mRNA. Approximately 2 μg of poly(A)-selected RNA prepared from two human cell lines (HPB-ALL [lane 1] and Raji [lane 2]) or human thymus tissue (lane 3) was loaded in each lane. The RNA was transferred to a nitrocellulose filter, and the filter was probed with a 32P-labeled probe prepared from Sip1 cDNA. The autoradiograph of the filter is shown.

FIG. 3
FIG. 3

Sequence similarities between Sip1 and SRp75 (A) and between Sip1 and Drosophila SWAP (B). Identical amino acid residues are in boldface and underlined. Conserved (I-L-V, D-E, K-R, S-T) amino acids are underlined.

FIG. 4
FIG. 4

Interactions between Sip1 and different RS domain-containing splicing factors (as indicated under the x axis) detected by the yeast two-hybrid interaction assay. Quantitative liquid β-galactosidase assays were performed with yeast extracts from at least three independent yeast isolates for each combination. Relative β-galactosidase activities shown represent fold activation above background (see Materials and Methods).

FIG. 5
FIG. 5

Interaction of Sip1 with U2AF65 but not U2AF35 as assayed by coimmunoprecipitation. Sip1, U2AF65, and U2AF35 were labeled with [35S]methionine by in vitro translation reactions. Individual proteins alone or proteins after coincubation were immunoprecipitated with anti-Sip1 antiserum (lanes 2 to 6). Lane 1 contains U2AF35 immunoprecipitated by anti-FLAG antibody, which recognizes the epitope tag on the in vitro-translated U2AF35. Lanes 2 and 4 contain immunoprecipitates from U2AF35 (lane 2) and U2AF65 (lane 4) in vitro translation products precipitated by anti-Sip1 antibody, showing that this antibody does not cross-react with either U2AF35 or U2AF65. Lanes 3 and 5 contain immunoprecipitates formed after coincubation of Sip1 with U2AF35 (lane 3) or with U2AF65 (lane 5), using anti-Sip1 antibody. Lane 6 contains Sip1 in vitro translation products precipitated by anti-Sip1.

FIG. 6
FIG. 6

The essential role of Sip1 in pre-mRNA splicing is not redundant with that of SC35, ASF/SF2, or U2AF65. (A) Sip1 protein in HeLa cell nuclear extract could be depleted by anti-Sip1 but not preimmune serum. Western blotting analysis was performed with the anti-Sip1 antibody, using HeLa nuclear extract (lane 1), the extract after immunodepletion with anti-Sip1 (lane 2) or preimmune serum (lane 3), or the purified recombinant Sip1 protein from the baculovirus expression system (lane 4). (B) Coomassie blue staining of the purified recombinant Sip1 protein (lane 1), U2AF65 protein (lane 2), and protein molecular mass markers (lane 3). (C) The recombinant Sip1, but not SC35, ASF/SF2, or U2AF65, could partially restore splicing activity of the Sip1-depleted nuclear extract. In vitro splicing of human β-globin pre-mRNA was carried out for 90 min with HeLa nuclear extract after immunodepletion by the preimmune serum (Mock depl., lane 1) or anti-Sip1 (lanes 2 to 10). The splicing extract was supplemented with 100 and 300 ng of purified Sip1 (lanes 3 and 4), SC35 (lanes 5 and 6), ASF/SF2 (lanes 7 and 8), or U2AF65 (lanes 9 and 10). The splicing reaction products were analyzed on a 6% polyacrylamide gel.

FIG. 7
FIG. 7

Sip1 interacts with U2AF65 in HeLa cell nuclear extract, and U2AF65 enhances splicing activity of the Sip1-reconstituted nuclear extract. (A) The level of U2AF65 in the Sip1-immunodepleted nuclear extracts was less than in the mock-depleted nuclear extracts as detected by Western blotting. Approximately 150 μg of nuclear extracts which had been immunodepleted by using either preimmune (lane 1) or anti-Sip1 (lane 2) serum was fractionated by SDS-PAGE (10% gel), transferred to a nitrocellulose membrane, and probed with anti-U2AF65 antibody (77). (B) U2AF65 further enhanced the splicing reconstitution by Sip1 in the Sip1-depleted nuclear extract. Lanes 1 to 9 contain the products of in vitro splicing of human β-globin pre-mRNA after a 30-min incubation; lanes 9 to 16 are the corresponding reaction products after a 90-min incubation. The splicing reaction products obtained with untreated HeLa nuclear extract (NE) are shown in lanes 8 and 16. The splicing products obtained with HeLa nuclear extract after immunodepletion by the preimmune serum (Mock depl.) are shown in lanes 1 and 9. Lanes 2 to 5 and 10 to 13 contain the splicing reaction products obtained with Sip1-immunodepleted HeLa nuclear extract supplemented with 0, 100, 200, and 300 ng of purified recombinant Sip1 protein after 30- or 90-min incubation. Lanes 6 and 7 and lanes 14 and 15 show the splicing products obtained with the Sip1-depleted extract supplemented with 200 ng of purified Sip1 and 100 or 200 ng of purified U2AF65 protein, respectively.

FIG. 8
FIG. 8

Involvement of Sip1 in spliceosome assembly. The splicing complexes were formed with 32P-labeled pPIP85.A pre-mRNA at different time points in the presence of ATP, using nuclear extracts which were immunodepleted (depl.) by using the preimmune or anti-Sip1 serum (lanes 1 to 4 and 5 to 8, respectively). The splicing complexes were separated on a native 4% polyacrylamide gel by electrophoresis. The autoradiograph of the gel is shown. Positions of A and B complexes and nonspecific H complex are indicated.

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