Identification and characterization of a novel serine-arginine-rich splicing regulatory protein

Abstract

We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

FIG. 1

Amino acid sequence of SRrp86. (A) The amino acid sequence of rat SRrp86 is shown with the RRM shaded and the conserved RNP-2 and RNP-1 boxes in italics. The RS domains are in white with a gray background, while a unique region rich in glutamic acid and lysine (EK-rich region) is in white with a black background. The human and rat proteins are 86% identical, with the exception of a 16-amino-acid insertion in the human protein. (B) SRrp86 was in vitro translated (IVT) in the presence of [³⁵S]methionine and subjected to SDS-PAGE. Molecular masses are in kilodaltons.

FIG. 2

SRrp86 inhibits SR proteins but is not required for splicing. (A) Affinity-purified antibodies against SRrp86 were used to probe a Western blot of nuclear extract (NE), S100 extract, and purified SR proteins. The asterisk denotes a 77-kDa protein that cross-reacts with anti-SRrp86 antibodies only under denaturing conditions. (B) Nuclear extracts were immunodepleted of SRrp86 and subjected to Western blot analysis using antibodies against SRrp86. Note that the cross-reactive 77-kDa protein did not coimmunoprecipitate. Molecular masses in kilodaltons are shown to the left of panels A and B. (C) In vitro splicing of an adenovirus-derived substrate was carried out in control extracts or in extracts immunodepleted of SRrp86 (ΔSRrp86 NE). (D) In vitro splicing of the same substrate as in panel C was performed in splicing-deficient S100 extracts in the absence (lane 2) or presence (lane 3) of SR proteins purified from calf thymus (0.5 μg). For comparison, splicing in nuclear extract is shown with the products and intermediates of splicing as indicated. Increasing amounts of SRrp86 (1.5 to 3 μg) inhibited complementation by calf thymus SR proteins (lanes 4 and 5), whereas addition of the ΔRS mutant (1.5 to 3 μg) did not (lanes 6 and 7), nor did the addition of recombinant ASF/SF2 (1.85 to 3.7 μg; lanes 8 and 9).

FIG. 3

Specificity of SRrp86 for individual SR proteins. (A) Individual recombinant SR proteins were used to complement S100 extract splicing of an adenovirus-derived substrate. SRrp86 (0.75 to 1.5 μg) or an equimolar amount of the ΔRS mutant was added to S100 extracts in the presence of the indicated recombinant SR protein. The first lane in panel A shows splicing in unsupplemented S100 extracts. (B) Rescue of splicing in S100 extracts by pairwise combinations of recombinant SR proteins in the presence of SRrp86 (1.5 μg) or the ΔRS mutant. In each set, a third SR protein (as indicated) was also added to control for possible nonspecific inhibition by excess recombinant protein. The concentrations of SRrp86, ΔRS, and recombinant SR proteins used in this panel were determined in separate titration experiments.

FIG. 4

Splice site selection by SR proteins affected by SRrp86. (A) In vitro splicing of a substrate derived from β-globin containing competing 5′ splice sites (5′D-16X) was performed in the presence of purified calf thymus SR proteins supplemented with the indicated amount of either WT SRrp86 or the ΔRS mutant. (B) Splicing of an α-TM-derived substrate containing competing 3′ splice sites was performed in the presence of purified calf thymus SR proteins supplemented as in panel A. (C) The 5′D-16X (left panel) and α-TM-derived substrates (right panel) were spliced in the presence of recombinant SR (rSR) protein ASF/SF2 (0.75 μg), SC35 (0.33 μg), SRp20 (0.6 μg), or SRp55 (1.1 μg) with or without SRrp86 (1.5 μg). As in Fig. 3B, the amounts of each of the proteins used in panel C were determined in separate titration experiments. The precursor and products of splicing for each substrate are diagrammed to the left of each gel.

FIG. 5

SRrp86 interacts with SR proteins. (A) Recombinant proteins were expressed in baculovirus-infected cells, purified by passage over Ni-NTA agarose, subjected to SDS-PAGE, and stained with Coomassie blue. Molecular masses in kilodaltons are shown on the left. (B) Recombinant proteins separated by SDS-PAGE as in panel A were transferred to PVDF membranes, and incubated with equivalent amounts of ³⁵S-labeled SRrp86 or the ³⁵S-labeled ΔRS mutant. (C) CNBr-activated Sepharose was coupled to WT SRrp86 or the ΔRS mutant or treated with ethanolamine (mock beads). Splicing extracts were incubated with the various resins, and flowthrough extracts were used in splicing reactions alone (lanes 1 to 3) or with the addition of purified SR proteins (lane 4). (D) Proteins retained on the resins in panel B were separated by SDS-PAGE, and Western blot analyses were performed with anti-SR antibody mAB1H4. The identities of known SR proteins are indicated on the left, and molecular masses are indicated on the right in kilodaltons. A longer exposure of the bottom portion of the gel shows the retention of SRp20.

FIG. 6

In vivo regulation of 5′ splice site selection by SRrp86. HeLa cells were transfected with the in vivo splicing construct pcDNA 5′D-16X (A) or adenovirus E1A (B). Cells were cotransfected with either a control vector (pcDNA) or a vector expressing SRrp86, the ΔRS mutant, or ASF/SF2, as indicated. RNA was isolated 48 h after transfection, and RT-PCR was performed to amplify the three possible spliced products, which were quantitated by PhosphorImager analysis, as indicated. The positions of the primers used for RT-PCR are indicated by the arrows.