Combinatorial control of signal-induced exon repression by hnRNP L and PSF - PubMed (original) (raw)

Combinatorial control of signal-induced exon repression by hnRNP L and PSF

Alexis A Melton et al. Mol Cell Biol. 2007 Oct.

Abstract

Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.

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Figures

FIG. 1.

FIG. 1.

Endogenous CD45 splicing regulation is reproduced in vitro. (A) Schematic representation of the wild-type (WT) and ΔESS1 exon 4 minigenes. Variable exon 4 of CD45 is flanked by constitutive exons 3 and 7 of CD45. Exons and introns are represented by boxes and lines, respectively. The ESS1 silencer element is represented by the crosshatched region within exon 4 of the WT minigene. This element has been removed and replaced with heterologous sequence from the CD45 exon 14 in the ΔESS1 minigene. (B) In vitro splicing of WT and ΔESS1 minigenes. Using an assay established previously (9, 27), capped, unlabeled RNA derived from the WT and ΔESS1 constructs was incubated with nuclear extract prepared from either the resting (R) or the stimulated (S) JSL1 cells. Splice products were then analyzed by RT-PCR (see Materials and Methods) to calculate the percentage of exon 4 (E4) inclusion. This quantification and all others in this study are derived from at least three independent experiments, with averages (bold) and standard deviations (small type) given.

FIG. 2.

FIG. 2.

Increased silencing activity of hnRNP L following stimulation accounts for a portion of the activation-induced skipping of the CD45 exon 4. (A) Analysis of hnRNP L by 2D electrophoresis. Resting or stimulated JSL1 NE was subjected to separation by 2D electrophoresis and blotted with hnRNP L antibody (see Materials and Methods). pH labels correspond to the pI predicted for proteins migrating at that position. (B) Functional evaluation of hnRNP L purified from either resting or stimulated NE. Equal amounts of purified hnRNP L from either resting or stimulated NE (see Fig. S3A in the supplemental material) were titrated into in vitro splicing reaction mixtures containing the WT CD45 minigene and NE prepared from resting JSL1 cells (as shown in Fig. 1). Purification was done by affinity to poly(CA) RNA as described by Hui et al. (10). Addition of 100 ng of recombinant GST-hnRNP L (Rec. lane) is shown as a positive control. (C) Quantitation of in vitro splicing assays as shown in panel B. All values were normalized to exon 4 (E4) inclusion in the absence of hnRNP L addition (set to 100% inclusion). Statistical analysis reveals that the difference between the activity of resting hnRNP L and that of stimulated hnRNP L is significant (P < 0.05). (D) Antisense knockdown of hnRNP L using morpholino oligonucleotides in JSL1 cells. Shown are RT-PCR analysis of the WT minigene RNA isolated from JSL1 cells treated with the indicated amount of the hnRNP L morpholino oligonucleotide (L-MO) under either resting (R) or stimulated (S) conditions and Western blotting analysis of hnRNP L and U1A (loading control) from protein samples isolated from the cells used for RT-PCR analysis. (E) Bar graph represents the quantitation of the RNA splicing analysis for the WT minigenes as well as results from parallel experiments with a minigene lacking the ESS1 sequence (ΔESS1).

FIG. 3.

FIG. 3.

PSF and p54nrb are added to the ESS1-binding complex following stimulation. (A) The table shows the proteins identified by mass spectrometry analysis of proteins isolated from resting or stimulated NE following RNA affinity purification with ESS1. + indicates the positive identification of multiple peptides by mass spectrometry and − indicates the absence of peptides as found by mass spectrometry. (B) Western blotting analysis of total nuclear protein (NE) or direct RNA affinity purifications from either resting or stimulated NE using either wild-type ESS1 RNA (ESS1), mutant RNA (Mut1; see Fig. 5A), or nonspecific control RNA (NS), using antibodies against hnRNP K, hnRNP D, hnRNP A1, and hnRNP A2 as indicated. (C) Western blotting analysis of PSF binding to wild-type ESS1 RNA from resting (R) or stimulated (S) NE. Equal concentrations of total NE were analyzed in parallel. (D) Western blotting analysis of direct RNA affinity purifications from resting or stimulated NE, as described in panel B, using antibodies to PSF, p54nrb, and hnRNP L (loading control) as indicated.

FIG. 4.

FIG. 4.

PSF increases ESS1-dependent silencing under stimulated conditions. (A) In vitro splicing of the wild-type ESS1 minigene in either resting or stimulated NE in the presence of the indicated amounts of monoclonal PSF antibody. (B) Quantitation of the CD45 exon 4 (WT E4) inclusion in in vitro splicing assays following the addition of PSF antibody. Levels of WT E4 inclusion were normalized to the level of E4 inclusion in the absence of antibody (set at 1.0) in the corresponding resting (solid line) or stimulated (dashed line) NE. Data are also shown for preincubation of PSF antibody with bacterially purified, inactive His-tagged PSF prior to addition into splicing reaction mixtures with stimulated (S) NE (gray line). (C) Quantitation of the effect of PSF antibody on the splicing of a minigene lacking the ESS1 sequence (ΔESS1) in resting or stimulated NE. Analysis was done as described in the legend to panel B. (D) Quantitation of the effect of hnRNP L and hnRNP A2/B1 antibody on the splicing of the WT minigene in resting or stimulated NE. Analysis was done as described in the legend to panel B.

FIG. 5.

FIG. 5.

PSF from resting and activated JSL1 cells exhibits differential silencing activity. (A) Schematic of the experimental outline for testing the effect of cellular stimulation on PSF activity (see Materials and Methods for details). Unlabeled ovals in the final step indicate the potential presence of coassociated proteins in our purification scheme that are below the level of detection as shown in panel B. Mock purification from stimulated extracts lacking Flag-PSF is used as a control for nonspecific proteins that could associate with the anti-Flag beads. (B) Anti-Flag Western blotting and the Coomassie-stained gel of protein isolated in the purifications are shown in panel A. The far left lane in the anti-Flag blot is the mock purification. PSF is not visible by silver stain despite its clear abundance as shown by Western blotting and Coomassie stain. In the Coomassie-stained gel, the second prominent band corresponds to anti-Flag antibody heavy chain (H.C.), which routinely gets stripped off the beads to some extent during the purification procedure. (C) In vitro splicing, with WT and ΔESS1 minigenes, as shown in Fig. 1. All reaction mixtures were done with extracts from resting cells not expressing Flag-PSF, either alone or supplemented with purification reaction mixtures shown in panel B. (D) Quantification of three independent experiments, as described in the legend to panel C. Exon inclusion in the extract-only lane is set to 100% in each case.

FIG. 6.

FIG. 6.

hnRNP L and PSF contribute independently to the signal-induced increase in ESS1 silencing. (A) The complete sequence of the 60-nucleotide ESS1 element is shown with the short and long copies of the ARS motif underlined. The base changes within the two mutants of ESS1 (Mut1 and Mut2) are shown. (B) Quantitation of in vitro splicing of WT (white), Mut1 (gray), and Mut2 (crosshatched) minigene RNA in resting extract (basal skipping) or a comparison of resting versus stimulated extracts (activation-induced skipping) is shown. Change (_n_-fold) is a measure of the difference in splicing between resting and activated conditions and is calculated as the (percentage of exon 4 [E4] inclusion/percentage of E4 exclusion)resting/(percentage of E4 inclusion/percentage of E4 exclusion)stimulated. (C) Western blotting analysis of hnRNP L and PSF in ESS1 affinity purifications from stimulated NE done in the absence or presence of a nonbiotinylated RNA competitor as indicated (ESS1, Mut1, Mut2, and NS). (D) Quantitation of the Western blots in panel C. Values were normalized to the amount of protein bound in the absence of competitor RNA (set to 100%).

FIG. 7.

FIG. 7.

Combined inhibition of hnRNP L and PSF abolishes activation-induced exon repression. (A) Quantitation of in vitro splicing of WT exon 4 minigene RNA in either resting (R) or stimulated (S) NE with antibodies to hnRNP L and PSF added alone or in combination. The inclusion of exon 4 (E4) was normalized relative to that observed in resting extracts in the absence of antibody (set at 100%). + represents the addition of 2 μg of hnRNP L antibody or 2 μg of PSF antibody. (B) Antisense knockdown of hnRNP L, PSF, or p54nrb, alone or in combination in JSL1 cells. Bar graph represents the level of exon skipping of the WT minigene in either resting or stimulated cells after treatment with the indicated morpholino oligonucleotide. Western blots of representative samples are shown with U1A as a loading control.

FIG. 8.

FIG. 8.

Model of the ESS1-binding complex under resting and stimulated conditions. In resting cells, the ESS1 complex is bound by at least five hnRNPs, with hnRNP L acting as the main functional regulator of basal exon silencing. Following cellular activation, the modification state of hnRNP L is altered, correlating to an increase in the silencing activity of the protein. Additionally, PSF is added to the complex and mediates a further increase in the repressive activity of the ESS1 element.

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