HnRNP L represses exon splicing via a regulated exonic splicing silencer - PubMed (original) (raw)

HnRNP L represses exon splicing via a regulated exonic splicing silencer

Caryn R Rothrock et al. EMBO J. 2005.

Abstract

Skipping of mammalian exons during pre-mRNA splicing is commonly mediated by the activity of exonic splicing silencers (ESSs). We have recently identified a regulated ESS within variable exon 4 of the CD45 gene, named ESS1, that is necessary and sufficient for partial exon repression in resting T cells and has additional silencing activity upon T-cell activation. In this study, we identify three heterogeneous nuclear ribonucleoproteins (hnRNPs) that bind specifically to ESS1. The binding of one of these proteins, hnRNP-L, is significantly decreased by mutations that disrupt both the basal and induced activities of ESS1. Recombinant hnRNP-L functions to repress exon inclusion in vitro in an ESS1-dependent manner. Moreover, depletion of hnRNP-L, either in vitro or in vivo, leads to increased exon inclusion. In contrast, the other ESS1-binding proteins, PTB and hnRNP E2, do not discriminate between wild-type and mutant ESS1 in binding studies, and do not specifically alter ESS1-dependent splicing in vitro. Together, these studies demonstrate that hnRNP-L is the primary protein through which CD45 exon 4 silencing is mediated by the regulatory sequence ESS1.

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Figures

Figure 1

Figure 1

ESS1 controls both basal and activation-induced regulation of CD45 exon 4. (A) A schematic of the CD45 gene and the five spliced isoforms of CD45 that are translated in humans, demonstrating the variable level of exon 4 inclusion in resting and activated T cells. (B) RT–PCR analysis of the splicing of three minigenes stably expressed in JSL1 cells under resting (−PMA) or stimulated (+PMA) conditions (see Materials and methods). These minigenes consist of introns and exons from the human β-globin gene (lines and white boxes), and either no insert (glo) or insertion of a WT (grey box, glo-ESS) or mutant (black box, glo-mESS) version of the ESS1-regulated silencer. The reactions for glo-ESS were overloaded on the gel as compared to glo and glo-mESS to allow for visualization of the three-exon product in these samples. Quantitation of inclusion of the central exon (% 3 exon) was achieved by averaging results from at least four independent clones and two independent experiments. The standard deviation for the averages shown is <10% in each case. (C) Sequence of the WT ESS1 RNA, functionally mutant ESS1 (mESS), and nonspecific (E14) RNAs used in all the experiments in this study. Bold nt's refer to the ARS consensus motif. Mutations in mESS relative to ESS1 are within the ARS motif and are indicated by plain underlined text.

Figure 2

Figure 2

An exon-repression complex specifically associates with ESS1. (A) 32P-labeled ESS1 RNA (0.1 pmol) was incubated in the presence (+) or absence (−) of nuclear extract from unstimulated JSL1 cells and resolved on a native gel to observe free and bound RNA species. Unlabeled competitor RNA was also added to the reactions as indicated. (B) In all, 1 fmol unlabeled, capped RNA derived from a minigene containing WT CD45 variable exon 4, flanked by constitutive exons 3 and 7, was incubated in nuclear extract from unstimulated JSL1 cells in the presence or absence of exogenous competitor RNA. The resulting spliced products were then assayed by RT–PCR, as done for RNA derived from cells. Quantitation of the % 3-exon product is the average of at least four experiments with a standard deviation of <20% for all values given. (C) RNA derived from a CD45 exon 4 minigene in which the ESS1 has been deleted and replaced with heterologous sequence was spliced in the same assay as used for (B). (D) In vitro splicing as in panel B of RNA derived from a CD45 exon 4 minigene in which the ESS1 has been mutated to the ‘mESS' sequence shown in Figure 1C.

Figure 3

Figure 3

HnRNPs L, E2, and PTB are identified as ESS1-associated proteins by RNA affinity purification. (A) Affinity purification of proteins associated with ESS1 RNA. Silver stained SDS–PAGE gel of proteins isolated from JSL1 nuclear extract by virtue of association with chemically synthesized, 5′-biotinylated ESS1, mESS, and E14 RNAs (see Materials and methods for details). (B) Western blot of SDS–PAGE gel from panel A with specified antibodies. (C) Sequence of ESS1 with predicted high-affinity binding sites indicated for hnRNP L (bold lines), PTB (dotted lines), and hnRNP E (plain lines).

Figure 4

Figure 4

Mutations that disrupt the function of ESS1 inhibit binding of HnRNP L. (A) HnRNP L and PTB crosslink to ESS1 RNA from nuclear extract. Nuclear extract was incubated under splicing conditions with approximately 0.1 pmol uniformly 32P-labeled ESS1 RNA, treated with UV light, digested with RNAses, and then either directly resolved on a 10% SDS–PAGE gel (NE lane) or immunoprecipitated with antibodies as specified (IP lanes), and the precipitates were resolved on the gel. (B) UV crosslinking experiment as in panel A, except that no immunoprecipitation was carried out and unlabeled competitor RNAs as indicated were added during the binding reaction. The indicated identities of the crosslink species are based on experiments as shown in panel A. (C) UV crosslinking experiments using approximately 0.05 pmol purified recombinant GST-hnRNP L, GST-PTB or MBP-hnRNP E, and 0.1 pmol 32P-labeled ESS1 either alone (0) or in the presence of additional unlabeled ESS, mESS, or E14 competitor, as indicated.

Figure 5

Figure 5

HnRNP L induces exon repression in in vitro splicing assays. (A) In vitro splicing assays with WT CD45 exon 4 minigene as described in Figure 2B. Splicing reactions contained either nuclear extract alone (−) or supplemented with purified recombinant GST-hnRNP L, GST-PTB, and/or MBP-hnRNP E2, as indicated. Quantitation of replicate experiments is given in panel C. (B) In vitro splicing as in panel A, but using the ΔESS splicing substrate that lacks the ESS1 regulatory sequence. Quantitation of replicate experiments is given in panel C. (C) Graphical representation of the three-exon product from at least four independent experiments, identical to those shown in panels A and B, and similar experiments with purified recombinant hnRNP A1. Numbers are given as % of three-exon product as compared to NE alone (set at 100%).

Figure 6

Figure 6

Depletion of hnRNP L in vitro or in vivo leads to increased inclusion of an ESS1-containing exon. (A) In vitro splicing assays lacking (−ESS) or containing (+ESS) exogenous ESS1 RNA similar to experiments shown in Figure 2B, in which purified recombinant GST-hnRNP L is also added to splicing reactions at the concentrations indicated. Quantitation is from at least four independent experiments, with a standard deviation of <20% of value. (B) In vitro splicing assays in the presence (+) or absence (−) of 2 μl 4D11 antibody and/or 100 ng recombinant GST-hnRNP L. Quantitation is derived from at least two independent experiments with standard deviation <10% of value. (C) RT–PCR analysis of WT or ΔESS minigene-derived RNA harvested from 293 cells transiently cotransfected with a minigene encoding vector and siRNAs against either GFP (G) or hnRNP L (L). Quantitation of three-exon product is derived from four independent transfections with standard deviation of <5%. (D) Western blot analysis of hnRNP L, or Erk1/2 as internal control, from transfections corresponding to those shown in panel C. The average reduction of hnRNP L in transfections with hnRNP L siRNAs versus GFP siRNAs was 50±10%.

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