A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4 - PubMed (original) (raw)

A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4

Justin D Topp et al. RNA. 2008 Oct.

Abstract

The human CD45 gene encodes a protein-tyrosine phosphatase that exhibits differential isoform expression in resting and activated T cells due to alternative splicing of three variable exons. Previously, we have used biochemical methods to identify two regulatory proteins, hnRNP L and PSF, which contribute to the activation-induced skipping of CD45 via the ESS1 regulatory element in variable exon 4. Here we report the identification of a third CD45 regulatory factor, hnRNP L-like (hnRNP LL), via a cell-based screen for clonal variants that exhibit an activation-like phenotype of CD45 splicing even under resting conditions. Microarray analysis of two splicing-altered clones revealed increased expression of hnRNP LL relative to wild-type cells. We further demonstrate that both the expression of hnRNP LL protein and its binding to ESS1 are up-regulated in wild-type cells upon activation. Forced overexpression of hnRNP LL in wild-type cells results in an increase in exon repression, while knock-down of hnRNP LL eliminates activation-induced exon skipping. Interestingly, analysis of the binding of hnRNP L and hnRNP LL to mutants of ESS1 reveals that these proteins have overlapping, but distinct binding requirements. Together, these data establish that hnRNP LL plays a critical and unique role in the signal-induced regulation of CD45 and demonstrate the utility of cell-based screens for the identification of novel splicing regulatory factors.

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Figures

FIGURE 1.

Dual reporter assay of CD45 splicing identified clones with “activated” splicing phenotype. (A) Scheme of dual-reporter system. (B) Flow cytometry analysis of GFP expression in mock-infected resting 3.14 cells (filled), initial cDNA infected cell population (line, left), or resting cell population after recovery from the initial sort (line, right). Circle on left indicates cells that were gated and collected in the initial sort. (C) Graphic representation of exon 4 inclusion in reporter minigene in 106 GFP+ clones (▲), compared with resting (●) or stimulated (■) 3.14 cells. Exon 4 inclusion in all cases was quantitated by low-cycle RT–PCR. Triangles corresponding to data from the three clones chosen for further study are indicated.

FIGURE 2.

Individual clones show activated pattern of CD45 splicing without up-regulation of general activation markers. (A) Flow cytometry analysis of GFP expression in isolated resting clones compared with that in resting (−PMA) or stimulated (+PMA) 3.14 cells. (B) RT–PCR analysis of splicing of the endogenous CD45 gene in 3.14 cells and clones. Quantitation of R0 isoform relative to total CD45 RNA is indicated. This and all other quantification in this study are derived from at least two to three independent experiments with deviations of <l15% of average value. (C) Flow cytometry analysis of CD45R0 expression in isolated clones compared with that in 3.14 cells, all under resting conditions. (D) Flow cytometry analysis of CD69 expression in clones versus 3.14 cells grown under resting or stimulated conditions.

FIGURE 3.

HnRNP LL is up-regulated in clones 39 and 88 as well as upon PMA stimulation of WT cells. (A) RT–PCR analysis of hnRNP LL mRNA at 20, 24, and 28 cycles, hnRNP L mRNA at 12, 16, and 20 cycles, or 16, 20, and 24 cycles for CD45 mRNA. Fold increase of signal relative to 3.14 resting cells shown is the average difference quantitated for at least the two lowest cycle points. Constitutive exons 8–10 from the endogenous CD45 gene are used as a control. (B) Western blot analysis of hnRNP LL, L, and PSF in nuclear extracts (NE) from 3.14 cells and clones grown under resting conditions, as well as from parental JSL1 cells grown under resting (−PMA) or activated (+PMA) conditions. All extracts were normalized for total protein level prior to loading and loaded at 5 or 15 μg of total protein per lane. Expression of LL, L, PSF, or U1A (loading control) were quantitated by densitometry at both loading points and averaged to give number shown.

FIGURE 4.

Binding of hnRNP LL to ESS1 increases relative to hnRNP L upon cellular stimulation. (A) Western blot of hnRNP L and LL following affinity purification (“pull-down”) with biotinylated ESS1, mutant (mESS) or nonspecific (NS) 60-mer RNAs from JSL1 nuclear extract (NE) prepared from resting (−PMA) or activated (+PMA) cells. (B) UV cross-linking experiments done with uniformly labeled ESS1 or mESS RNA and nuclear extract from resting or stimulated cells. As indicated, parallel cross-linked samples were subject to immunoprecipitation with antibodies to hnRNP L, LL, or an IgG control prior to running of the SDS-PAGE.

FIGURE 5.

HnRNP LL functionally contributes to repression of CD45 exon 4. (A) Schematic of the WT ESS1 exon 4 minigene described previously (Melton et al. 2007) used in Figures 5 and 6. (B) RT–PCR of WT minigene expression following transient cotransfection in JSL1 cells with constructs expressing Flag-hnRNP L, Flag-hnRNP LL, or Flag vector alone. Percent exon 4 skipping is calculated from two to three independent experiments. Standard error in each case is <15% of exon-skipped value. (*) Cryptic product observed variably with reduced cell viability. (C) Total protein extracts from transfections in B blotted for Flag, hnRNP LL, or hnRNP L. (*) The band corresponding to hnRNP LL, lower band is a nonspecific signal observed on occasional blots. (D) RT–PCR of WT minigene expression following transient cotransfection with morpholino oligomers (MO) that block translation of hnRNP L or LL. Percent exon 4 skipping is calculated as in B. (E) Total protein extracts from transfections in D blotted for hnRNP L or LL. (*) The band corresponding to hnRNP LL.

FIGURE 6.

Specificity of hnRNP LL binding to ESS1 is consistent with a unique role in signal-induced exon repression. (A) Sequence of WT ESS1 with location and identity of mutations indicated. Underlined residues correspond to the ARS core motif repeats. (B) UV cross-linking and immunoprecipitation as done in Figure 4 for WT and mutant ESS1 RNAs. (C) RT–PCR analysis of minigenes with WT or indicated mutant ESS1 sequence inserted into ESS1 location of construct shown in Figure 5A. Minigenes were cotransfected with 1 or 3 μg of Flag-hnRNP LL, and quantitation of RT–PCR was as done for Figure 5. Overall exon skipping was somewhat lower in these experiments than in Figure 5 due to inherent variation in transient transfections, but data shown in each figure are from parallel experiments and are thus internally controlled.

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A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4 - PubMed (original) (raw)