HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing - PubMed (original) (raw)

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

Marco Preussner et al. Nucleic Acids Res. 2012 Jul.

Abstract

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4-6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4-6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons.

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Figures

Figure 1.

Figure 1.

HnRNP LL is differentially expressed in B-cell lines and regulates CD45 alternative splicing. (A) High LL expression correlates with the plasma cell stage of B-cell development, while L is expressed at similar levels in most of the B-cell lines tested (names of cell lines above the lanes). Lysates from B-cell lines derived from early, mature or activated stages of B-cell development [left panel: pre B-cell stage (pre), lymphoblastoid cell lines (LCL) and Burkitt lymphoma B-cell lines (BL), as indicated below] and from plasma cell-derived B-cell lines (right panel: myeloma cell lines) were analyzed by western blot for L/LL (GAPDH as a loading control). (B) CD45 alternative exon repression correlates with LL expression levels. CD45 alternative splicing analysis by RT-PCR, comparing two different B-cell lines (Burkitt lymphoma line DG75, myeloma cell line U-266). Primer pairs (CD45 ex3–9, 3–5 and 5–7) and products (CD45 splice forms R456, R56, R5, R0; exons 4 and 6 inclusion/skipping forms; constitutive ex8–9 product) are labeled on the side. M, DNA size markers. Below, the expression of LL, L and γ-tubulin were detected by western blotting. (C) HnRNP LL overexpression is sufficient for CD45 exons 4–6 skipping and the generation of the R0 form. DG75 cells were transfected with increasing amounts of LL-V5-His plasmid (0.05, 0.2, 0.5 or 2 µg), or as a control, GFP (1 µg). Total RNA was analyzed by RT-PCR (top) for CD45 alternative splicing [products and primer pairs indicated on the right, as in panel (B)]. M, DNA size markers. Below, LL overexpression was assessed by western blotting, with GAPDH as a control.

Figure 2.

Figure 2.

RNA-Seq analysis of CD45 alternative splicing in DG75 B-cells. LL (or as a control, GFP) was overexpressed in DG75 B-cells, followed by RNA-Seq analysis of total RNA. On the top, the read densities across the CD45 exons 3–7 region are represented, comparing GFP control (in blue) and LL overexpression (in red). The numbers below indicate the respective read densities of exons 3–7. In the middle, the ratios of LL/GFP exon read densities, normalized to the constitutive exons 3 and 7, are given, below, the exon–intron structure. The bottom part summarizes the absolute junction read counts, with the arrows indicating changes in a particular splice event after LL overexpression (↑ increase; ↓ decrease).

Figure 3.

Figure 3.

CD45 alternative splicing of a minigene in HeLa cells: specific repression of CD45 alternative exons 4–6 by hnRNP L and LL. (A) L (ΔL), LL (ΔLL), or both of them (ΔL + ΔLL) were RNAi-downregulated in HeLa cells, including a control luciferase knockdown (Δluc), as confirmed by western blot for L, LL and GAPDH. (B) Following knockdown, the CD45 WT minigene was transfected, and CD45 alternative splicing was assayed by RT-PCR (primer pairs indicated above the lanes, and schematically shown below). The RT-PCR products indicative of the R456, R45, R5 and R0 forms (for primer pair 3–7), or for the inclusion/skipping-products (for primer pairs 5–7, 3–5 and 4–6; see brackets) are marked on the left. All lanes come from a single gel, but were rearranged for clarity. M, DNA size markers. Below, the repressor activities of L versus LL for CD45 exons 4–6, as well as for the entire exons 4–6 unit are summarized (++ strong; + weak; − not significant).

Figure 4.

Figure 4.

Mutational analysis of CD45 silencer elements in exons 4 and 6. (A) CD45 WT minigenes and mutant derivatives [as indicated above the lanes; see panel (B) for schematic representations] were transfected in HeLa cells, followed by RT-PCR with primers against CD45 exons 3 and 7 (products represented on the right). For CD45 WT and mutants 4/5′CA, 6/m + 3′CA and 4/5′CA + 6/m + 3′CA, additional cotransfections were done with GFP (as control), with FLAG-L or FLAG-LL (as indicated above the lanes), followed by RT-PCR assays with exons 3–7 or 4–6 primer pairs (as indicated). The asterisk marks an unspecific PCR product, which could not be identified. M, DNA size markers. (B) Summary of CD45 mutant minigenes used (the position of mutations marked by thick vertical lines). The mutated nucleotide positions in exons 4 and 6 are specified below, in each case with the CA elements in bold letters and the mutations underlined. Spliced CD45 isoforms are represented on the right side, with single and double arrows indicating changes compared to wild-type.

Figure 5.

Figure 5.

In vitro binding of hnRNP L and LL to CD45 exons 4–6. (A) RNA binding of hnRNP L/LL correlates with repressor activity. Equimolar amounts of 3′-biotinylated RNAs, containing wild-type or mutant versions of each variable exon (as indicated above the lanes and schematically on the right), were prebound to Neutravidin agarose, followed by incubation in HeLa nuclear extract, washing at 100 mM KCl, release of bound protein and western-blot analysis for LL, L and, as a control, GAPDH. For comparison, nuclear extract input was analyzed in parallel (lanes 1; 20% for LL and GAPDH; 5% for L). Quantitation of L/LL pulldown is given below each lane (in % relative to the input, including SDs). (B) An RNA containing CD45 exons 4-5-6 in cis stabilizes hnRNP L binding, depending on exons 4 and 6. As described in panel (A), pre-mRNAs, containing either the wild-type sequence (exons 4-5-6) or a mutant derivative, combining silencer mutations in exons 4 and 6 (exons 4*-5-6*), were compared with single-exons 5 and 6 RNAs for L versus LL binding in nuclear extract (lanes 1–14). Complex stability was tested by washing under different salt conditions (as indicated on top: 100–300 mM KCl). In addition, L/LL binding to mutant exon 4-5-6 derivatives were compared, in which one of the three exons was replaced by DUP sequence (DUP-ex4, -ex5 and -ex6; lanes 15–18).

Figure 6.

Figure 6.

CD45 RNA-dependent interaction of hnRNP L and LL in vivo. (A) Schematic of CD45 minigenes and experimental protocol. (B) Interaction depends on CD45 RNA expression: HeLa cells were transfected with either FLAG-LL alone (lanes 1 and 2), or in combination with the CD45 WT minigene (lanes 3–6). As controls, CD45 WT was cotransfected with GFP (lanes 7–9), or FLAG-LL with GFP (lanes 10–12). Lysates were prepared without or with RNase treatment (as indicated), followed by FLAG pulldown assays. Coprecipitated L was detected by western blot, comparing input (lanes i; 2.5%) and bound material (lanes b; 20%), in parallel with LL, FLAG tag and loading control GAPDH. (C) Interaction depends on exons 4 and 6: Additional FLAG pulldown assays were performed after cotransfections of FLAG-LL and CD45 WT (lanes 13 and 14) or mutant minigenes carrying DUP-substituted exons 4–6 (DUP-ex4, -ex5, -ex6; lanes 15–20).

Figure 7.

Figure 7.

Model of CD45 alternative splicing of variable exons 4/5/6, determined by combined activities of L/LL paralogs (for details, see ‘Discussion’ section).

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