hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells

Abstract

The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.

FIG. 1

Isolation of p58 by Mono Q chromatography. (A) The ASP40 fraction from the WERI nuclear extract was loaded on a Mono Q column. Proteins were eluted by a linear KCl gradient from 0 to 0.5 M, and the UV absorbance at 280 nm was determined. The flowthrough (FT) is indicated with a solid bar, and the peak fraction containing the bulk of the p58 is shaded. (B) SDS-PAGE analysis of the Mono Q fractions. Proteins from the WERI ASP40 fraction (lane 1), the flowthrough fraction (lane 2), the peak fraction eluting at 0.2 M (lane 3), the 0.2 to 0.3 M fraction (lane 4), the 0.3 to 0.4 M fraction (lane 5), and the 0.4 to 0.5 M fraction (lane 6) were UV cross-linked to radiolabeled DCS RNA. These samples were then RNase treated, separated by SDS-PAGE, and detected by autoradiography.

FIG. 2

The p58 protein is immunoprecipitable with anti-Sm monoclonal antibody Y12. (A) Immunoprecipitation of proteins UV cross-linked to the DCS RNA. The WERI ASP40 fraction was UV cross-linked with radiolabeled DCS RNA and then subjected to RNase digestion. The mixture was immunoprecipitated with anti-Sm monoclonal antibody Y12 (lane 2), αTMG (lane 3), anti-SR protein antibody 16H3 (αSR; lane 4), and anti-hnRNP F antibody (αF; lane 5). The total cross-linked proteins are shown in lane 1. The immunoprecipitated radiolabeled proteins were separated by SDS-PAGE and detected with a PhosphorImager (Molecular Dynamics). (B) SDS-PAGE of the p58 protein isolated by anti-Sm immunoaffinity column. An immunoaffinity column was prepared by using the anti-Sm monoclonal antibody Y12. The Mono Q peak fraction was incubated with the Y12 beads. After the column was washed with buffer DG, the proteins were eluted with 2% SDS. Lane 1, nuclear extract; lane 2, peak fraction after Mono Q chromatography; lane 3, 2% SDS-eluted proteins from the anti-Sm column. Proteins were resolved by SDS-PAGE on a 10% gel in the absence of β-mercaptoethanol and stained with Coomassie blue. The p58 protein band is indicated with an arrow. This band was cut out and subjected to in-gel tryptic digestion, and the resulting peptides were separated by reverse-phase HPLC and sequenced by automated Edman degradation.

FIG. 3

Antibody to the C-terminal peptide sequence of hnRNP H reacts with hnRNP H but not hnRNP F. Shown is Western blot analysis of WERI nuclear extract with preimmune serum (lane 1), C-terminal peptide antibodies specific to hnRNP H (lane 2), and anti-hnRNP F antibodies (lane 3).

FIG. 4

hnRNP H is a component of the DCS complex. (A) Immunoprecipitation of the p58 protein cross-linked to the DCS RNA by anti-hnRNP H antibody. The ASP40 fraction of the WERI nuclear extract was UV cross-linked to radiolabeled DCS RNA and then subjected to RNase digestion. The mixture (lane 1) was immunoprecipitated with preimmune serum (lane 2), anti-hnRNP H antibody (lane 3), and anti-KSRP antibody (lane 4), followed by SDS-PAGE and autoradiography. (B) Supershift of the DCS complex by anti-hnRNP H antibody. The radiolabeled DCS RNA was incubated with the ASP40 fraction of the HeLa (lane 1) or WERI (lanes 2 to 4) nuclear extract. The WERI fraction was preincubated with nothing (lane 2), preimmune serum (lane 3), or anti-hnRNP H serum (lane 4). After incubation with protein, the DCS RNA was analyzed on a native electrophoretic gel. The free probe and the previously characterized DCS complex are indicated at the right.

FIG. 5

Anti-hnRNP H antibody immunoprecipitates src pre-mRNA splicing complexes from the in vitro splicing reaction. WERI nuclear extract was incubated with ³²P-labeled src BS7 (lanes 1 to 5) or Ad (lanes 6 to 10) pre-mRNA substrate under splicing conditions. Reaction mixtures were aliquoted to protein A-Sepharose beads carrying no antibody (Ab) (lanes 2 and 7), anti-Sm antibody (lanes 3 and 8), preimmune serum for the anti-hnRNP H antibody (lanes 4 and 9), or anti-hnRNP H antibody (lanes 5 and 10), and the RNAs were recovered after immunoprecipitation. RNAs extracted from the total splicing reaction are in lanes 1 and 6. These lanes contain RNA from one-fourth of the amount of extract used in the immunoprecipitations. RNAs were analyzed by electrophoresis on an 8% polyacrylamide gel containing 8 M urea.

FIG. 6

(A) Western blot analysis of different amounts of mock-depleted (lanes 1, 3, and 5) or hnRNP H-depleted (lanes 2, 4, and 6) WERI nuclear extract (NE) probed with anti-hnRNP H and anti-hnRNP F antibodies (top) or anti-KSRP antibody (bottom). (B) SDS-PAGE of recombinant hnRNPs H (lane 1) and F (lane 2) stained with Coomassie blue. N-terminal histidine-tagged hnRNPs H and F were expressed in E. coli in an inducible T7 RNA polymerase-based system. After overexpression and cell lysis, protein was dissolved in 6 M urea, loaded on a Ni-NTA column, and refolded in a linear gradient of 6 to 1 M urea. The renaturated protein was then eluted in 0.25 M imidazole.

FIG. 7

hnRNP H restores src pre-mRNA splicing in hnRNP H-depleted extract. (A) Splicing of the src BS7 pre-mRNA. In a 25-μl volume, reaction mixtures contained 6 μl of mock-depleted extract (M; lane 1), hnRNP H-depleted extract (D; lane 2), or hnRNP H-depleted extract supplemented with increasing amounts (0.1, 0.5, and 1 μg) of recombinant hnRNP H (lanes 3 to 5). (B) Splicing of the Ad pre-mRNA in mock-depleted extract (lane 1), hnRNP H-depleted extract (lane 2), or hnRNP H-depleted extract supplemented with increasing amounts (0.1, 0.5, and 1 μg) of recombinant hnRNP H (lanes 3 to 5). (C) Quantitation of hnRNP H depletion-reconstitution levels of src BS7 RNA species shown in panel A. (D) Splicing of the src BS7 pre-mRNA in mock-depleted extract (lane 1), hnRNP H-depleted extract (lane 2), or hnRNP H-depleted extract supplemented with increasing amounts (0.1, 0.5, and 1 μg) of recombinant hnRNP F (lanes 3 to 5).

FIG. 8

hnRNP H interacts with hnRNP F. (A) Recombinant hnRNP F and KSRP were translated in a rabbit reticulocyte lysate and labeled with [³⁵S]methionine (lanes 7 and 8). Radiolabeled hnRNP F and KSRP were incubated with histidine-tagged recombinant hnRNP H bound to Ni-NTA agarose beads. After incubation, beads were either treated with RNase A (lanes 3 and 6) or not treated (lanes 2 and 5). As negative controls, ³⁵S-labeled hnRNP F and KSRP were incubated with Ni-NTA beads in the absence of hnRNP H (lanes 1 and 4). (B) Coimmunoprecipitation of hnRNP F with hnRNP H by anti-hnRNP H antibody. Radiolabeled hnRNP F (³⁵S-F) was incubated with anti-hnRNP H-coupled protein A beads in the absence (lane 1) or presence (lane 2) of hnRNP H. Radiolabeled luciferase (³⁵S-LC) was used as negative control (lane 4). After washing, the proteins were recovered from the beads in 2% SDS and resolved by SDS-PAGE.