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HnRNP A1 tethers KSRP to an exon splicing silencer that inhibits an erythroid-specific splicing event in PU.1-induced erythroleukemia - PubMed

  • ️Thu Jan 01 2015

. 2015 Mar 15;5(4):1410-22.

eCollection 2015.

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HnRNP A1 tethers KSRP to an exon splicing silencer that inhibits an erythroid-specific splicing event in PU.1-induced erythroleukemia

Alexandre Douablin et al. Am J Cancer Res. 2015.

Abstract

Exon 16 inclusion is a critical splicing event that triggers the production of a functional protein 4.1R in mature normal erythroblasts, and is obviated in PU.1-induced erythroleukemia cells. Exon 16 contains an exonic splicing silencer (ESS16) that interacts with hnRNP A/B in heterologous cell context. We here show that ESS16 promotes the recruitment of a protein complex containing hnRNP A1 and a 79-kDa protein in nuclear extracts from either proliferative erythroleukemia cells or cells induced to terminal differentiation. By using 2D gel fractionation and mass spectrometry, we unambiguously identified KSRP as the 79-kDa component interacting with ESS16. Furthermore, we show that KSRP slightly decreases in erythroleukemia cells induced to terminal erythroid differentiation. Yet, KSRP inducible knockdown, through stable transfection of small hairpin KSRP RNA, did not alter exon 16 splicing, suggesting that KSRP alone does not modulate the splicing event. Interestingly, absence of hnRNP A1 prevented KSRP from binding to ESS16. Reciprocally, KSRP interaction with ESS16 was recovered when hnRNP A1 expression is restored in hnRNP A1-null cells. Collectively, this study establishes that hnRNPA1 is part of a KSRP-containing RNP complex, and emphasizes that, aside from its function in AU-rich element-mediated mRNA decay and its role in microRNA biogenesis, KSRP associates with hnRNP A1 to bind an ESS. These findings further support the role of members of the KH-domain protein family in organizing large RNA-protein complex formation, rather than primarily in modulating specific splicing events.

Keywords: Alternative splicing; KSRP; PU.1; cell differentiation; erythroleukemia; hnRNP.

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Figures

Figure 1
Figure 1

ESS16 binds to a 79 kDa nuclear factor in proliferating and differentiating erythroleukemia cell nuclear extracts. A. ESS16 32P-labeled RNA probe was in vitro transcribed under the control of SP6 promoter. B. EMSA using labeled ESS16 probe and nuclear extracts from proliferating (-) cells, or cells induced to erythroid differentiation by DMSO (+). 1, 2 and 3 indicate the 3 major complexes revealed. A control sample (Ctr), where the RNA was incubated in the absence of protein extract, shows only free labeled molecules. C. UV crosslinking experiments using 32P-labeled ESS16 RNA and nuclear extracts from proliferating (-) and DMSO-treated (+) 745A cells. The star points to the major crosslinked protein. Size markers are depicted on the right.

Figure 2
Figure 2

2-D electrophoretic analysis of ESS16-associated proteins. UV crosslinking assay was performed at an analytical (A, B) and preparative (C) scales, using 32P-labeled (A, B) or unlabeled (C) in vitro transcribed ESS16 RNA probes. A: SDS-PAGE showing the crosslinked proteins including the major 79 kDa component. (B, C) 2-D gel electrophoresis. Proteins were separated by IEF on immobilized pH gradients 3-10 in the first dimension. They were subsequently separated by SDS-PAGE in the second dimension. All three spots fractionated by SDS-PAGE at the 79 kDa position were analyzed by mass spectrometry.

Figure 3
Figure 3

KSRP expression during erythroid differentiation. A. Induction of erythroid differentiation results in decreased total KSRP expression. KSRP expression was analyzed by immunoblotting in untreated (-) cells or cells treated with DMSO (+), 10 μM (10) or 20 μM (20) of LY294002 (LY29) inhibitor. Actin served as a protein quality and sample loading standard. B. KSRP nuclear expression decreases in DMSO-treated cells. Fifteen μg of nuclear protein extracts were loaded on SDS-PAGE and immunoblotted using anti-KSRP antibody. Actin served as a protein quality standard. Nuclear extracts were prepared from HeLa cells, and MEL cells (clone 745 A) before and after DMSO exposure.

Figure 4
Figure 4

KSRP knockdown does not alter exon 16 erythroid splicing. A. Immunoblotting using anti-KSRP antibody to assess KSRP expression in cells stably transfected with shKSRP, as compared with control cells transfected with the scrambled shRNA (shCtr). Actin immunoblotting served as a loading control. Cells were analyzed in the absence (-) or presence (+) of doxycycline (dox), and before (-) or after (+) DMSO induction to differentiation. B. Exon 16 splicing efficiency was analyzed in shCtr and shKSRP cells in the same culture conditions described in A. Samples without RNA template (No RNA) or without adding reverse transcriptase (No RT) were run as RT-PCR controls. C. Semi-quantitative analysis of exon 16 splicing before and 5 days after DMSO (+DMSO) treatment. Cells were either uninduced (-dox) or induced (+dox) to express shKSRP RNA by adding doxycycline to the culture media. The data correspond to 3 different experiments; they are shown as mean ± SD.

Figure 5
Figure 5

KSRP knockdown does not alter hnRNP A1 expression in erythroleukemia cells. HnRNP A1 and KSRP expression was assessed by western blot. Cells were stably transfected with shKSRP or with the scrambled shRNA (shCtr), the expression of which is directed upon addition of doxycycline (dox). (+) and (-) denote the presence or absence of doxycycline, respectively. Actin immunoblot served as quality and loading control.

Figure 6
Figure 6

KSRP recruitment to ESS16 requires hnRNP A1. A. Immunoblot analysis of erythroleukemia cell clones using anti-hnRNP A1 specific antibody. The 745A clone serves as a control. It corresponds to the clone used in most experiments described in this work. CB3 is an uninducible clone that does not express hnRNP A1, due to the insertion of F-MuLV at Fli-2 site. CB3-A1 are CB3-derived clones, where hnRNP A1 cDNA was stably transfected. Several clones were analyzed. Two of them are shown: Cl.21 and Cl.36. This latter was used in subsequent experiments. Anti-actin antibody was used as quality and loading control of SDS-PAGE fractionated proteins. B. EMSA using ESS16 substrate and nuclear extracts obtained from CB3 cells (lane 2), CB3-A1 cells (lane 3), proliferating 745A cells (lane 4) or DMSO-treated 745A cells (lane 5). Lane 1: labeled RNA without nuclear extracts. The arrowhead points to the disruption of complex 1. C. UV crosslinking using ESS16 probe and nuclear extracts obtained from CB3 cells (lane 2), CB3-A1 cells, Cl36 (lane 3), proliferative (lane 4), or differentiating (lane 5) 745A cells. Size markers are indicated on the right. The star points to KSRP position.

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