A novel SR-related protein is required for the second step of Pre-mRNA splicing

Abstract

The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF65-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF35 in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.

FIG. 1.

(A) Amino acid sequence of mouse wild-type SRrp53 is shown. The RS domain is in white with a black background. (B) Human SRrp53 is shown aligned by the ClustalW method with the corresponding proteins from Mus musculus (mm), Rattus norvegicus (rat), Gallus gallus (chicken [gg]), X. laevis (xl), and Oryzias latipes (medaka fish [ol]). Identical residues are shown on a black background, residues conserved in 80% of the analyzed sequences are shown in a dark grey background, and residues conserved in 60% of the analyzed sequences are shown in a light grey background. Gaps are shown by dashed lines.

FIG. 2.

Characterization of SRrp53 by Western blot analysis. (A) The amounts of 10 μg of nuclear extract (lane 1), 10 μg of CIAP-treated nuclear extract (lane 2), and 50 μg of S100 extract (lane 3) were analyzed by Western blotting with anti-SRrp53-B4 (fourth bleed) antibody. Protein size markers (molecular weights [MW]) are indicated to the left. (B) The amounts of 20 ng of recombinant His-hnRNP L purified form baculovirus-infected Sf9 cells (lane 1) and 20 ng of T7-SRrp53 purified from 293T cells (lane 2) were analyzed as described for panel A. Protein size markers are indicated to the left.

FIG. 3.

(A) HeLa cells were transfected with a plasmid encoding GFP-SRrp53 and fixed 24 h posttransfection. Endogenous SC35 was detected by indirect immunofluorescence with an anti-SC35 MAb and fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Images were superimposed to reveal sites of overlap in yellow (Merge). (B) Effect of transcription inhibition on the subcellular localization of SRrp53. HeLa cells were transfected with a plasmid encoding T7-SRrp53; cells were then incubated with actinomycin D (+AcTD) plus cycloheximide for 3 h and fixed at 24 h posttransfection. The localization of the expressed proteins was determined by indirect immunofluorescence with anti-T7 MAb and FITC-conjugated secondary antibody. (C) Analysis of nucleocytoplasmic shuttling of SRrp53 by transient expression in interspecies heterokaryons. The SRrp53 protein was transiently expressed in HeLa cells, and 24 h posttransfection, the cells were treated with cycloheximide and subsequently fused with mouse NIH 3T3 cells in the presence of polyethylene glycol to form heterokaryons. The cells were further incubated for 2 h in the presence of cycloheximide, followed by fixation. The localization of the epitope-tagged proteins in the heterokaryons was determined by indirect immunofluorescence with anti-T7 MAb and FITC-conjugated secondary antibody (left panel). The cells were simultaneously incubated with DAPI for differential staining of human and mouse nuclei within heterokaryons (center panel). The arrows indicate the mouse nuclei within human-mouse heterokaryons. Phase-contrast images of the same heterokaryons are shown (right panel).

FIG. 4.

SRrp53 interacts with RS-domain-containing proteins in a yeast two-hybrid system. (A) Reconstitution of two-hybrid interactions found in a yeast two-hybrid screen. The center panel shows growth on selective media without adenine, and the right panel shows activation of the MEL1 reporter. In each panel, yeast cells were cotransformed with each of the prey plasmids (pGAD) isolated from the screen (indicated above the gels) and bait vectors expressing either full-length SRrp53 (first row), the N-terminal half of the protein comprising the RS domain (second row), the C-terminal half of the protein (third row), or the empty vector (fourth row). (B) A diagram illustrating the structure of each interactor. The portions of each protein covered by selected clones are indicated by bars (with the N- and C-terminal amino acid numbers shown). (C) Interactions between wild-type or mutant versions of SRrp53 and different splicing factors. Yeast cells were cotransformed with prey plasmids encoding different splicing factors (indicated above) with bait vectors expressing either full-length SRrp53 (first row), the N-terminal half of the protein comprising the RS domain (second row), the C-terminal half of the protein (third row), or the empty vector (fourth row) and were tested for their ability to grow in the absence of adenine. In all cases, activation of the ADE3 reporter correlated with activation of the MEL1 reporter (data not shown).

FIG. 5.

SRrp53 interacts with splicing factors in cultured mammalian cells. (A) IP assays with T7-hLuc7a are shown. Extracts prepared from 293T cells either transiently transfected with a plasmid encoding T7-hLuc7a (lanes 2 and 3) or mock transfected (lane 4) were incubated with anti-T7 antibody bound to Sepharose beads (Novagen). The bound proteins were separated on an SDS-10% polyacrylamide gel and analyzed by Western blotting with anti-SRrp53B4 antibody. Alternatively, the immunoprecipitate was treated with RNase before loading on the gel (lane 3). Lane 1 was loaded with 2% of the amount of extract used for each IP. (B) Extracts prepared from 293T cells were incubated with either anti-SRrp53B4 antibody (lanes 2 and 3) or preimmune serum (lane 4) bound to Sepharose beads and analyzed as described for panel A. The blot was probed with MAb 96 antibody, which recognizes SF2/ASF (25). Alternatively, the immunoprecipitate was treated with RNase before loading on the gel (lane 3). Lane 1 was loaded with 2% of the amount of extract used for each IP. (C) The interaction between SRrp53 and HCC1 was analyzed as described for panel B. The blot was probed with a polyclonal antibody raised in sheep, which specifically recognizes HCC1.

FIG. 6.

Effect of SRrp53 immunodepletion on pre-mRNA splicing in vitro. (A) The extent of SRrp53 depletion from HeLa nuclear extracts (NE) was assayed by Western blot analyses with an anti-SRrp53 antibody in serial dilutions of untreated extracts (lanes 1 through 5), extracts depleted with anti-SRrp53B4 antibody (lane 6), or preimmune-serum-depleted extracts (lane 7) (left panel). The profile of SR proteins was not affected by SRrp53 immunodepletion (right panel). (B) Effect of immunodepletion of SRrp53 on pre-mRNA splicing is shown. AdML pre-mRNA (lanes 1 and 2) or Ftz pre-mRNA (lanes 3 and 4) was incubated in depleted (lanes 1 and 3) or mock-depleted (lanes 2 and 4) nuclear extracts. Positions of splicing intermediates and products are indicated to the right. (C) Ftz pre-mRNA substrate was incubated in mock-depleted (lane 1) or SRrp53-depleted (lanes 2 through 4) nuclear extracts complemented with buffer (lanes 1 and 2) or 100 ng (lane 3) or 300 ng (lane 4) of T7-SRrp53 (left panel). Positions of splicing intermediates and products are indicated to the right. Ftz pre-mRNA substrate was incubated in mock-depleted (lane 1) or SRrp53-depleted (lanes 2 through 5) nuclear extracts complemented with buffer (lanes 1 and 2), hnRNP L (lane 3), SF2/ASF (lane 4), or SRp20 (lane 5) (right panel).

FIG. 7.

SRrp53 can regulate 5′ splice site selection in vivo. In vivo splicing analyses were performed with HeLa cells transiently cotransfected with an adenovirus E1A reporter plasmid and expression plasmids encoding for either mouse SRrp53, hnRNP A1 or SF2/ASF. (A) Diagram of the E1A reporter gene is shown. The alternative 5′ splice sites and splicing events that generates 13S, 12S, and 9S mRNAs are shown schematically. The location of the exon primers used for RT-PCR analysis is shown. (B) HeLa cells were transiently cotransfected with the adenovirus E1A reporter plasmid and the expression constructs for each of the proteins indicated above the gel or the parental plasmid (Control). RNA was harvested at 24 h posttransfection and analyzed by RT-PCR with a labeled forward primer, denaturing PAGE, and autoradiography as described in Materials and Methods. The positions of the unspliced pre-mRNA and of 13S, 12S, and 9S spliced mRNAs are indicated to the right. The 10S and 11S isoforms (*) did not arise from competition between alternative 5′ splice sites. (C) Quantitation of E1A mRNA isoforms in transfected cells is shown. The relative amounts of 13S, 12S, and 9S E1A mRNAs were calculated from the data given in panel B by using a phosphorimager, and the percentage of each isoform is shown. Each experiment was repeated four times, and the data represent averages, with bars indicating standard errors.