Autoregulation of Fox protein expression to produce dominant negative splicing factors - PubMed (original) (raw)
Autoregulation of Fox protein expression to produce dominant negative splicing factors
Andrey Damianov et al. RNA. 2010 Feb.
Abstract
The Fox proteins are a family of regulators that control the alternative splicing of many exons in neurons, muscle, and other tissues. Each of the three mammalian paralogs, Fox-1 (A2BP1), Fox-2 (RBM9), and Fox-3 (HRNBP3), produces proteins with a single RNA-binding domain (RRM) flanked by N- and C-terminal domains that are highly diversified through the use of alternative promoters and alternative splicing patterns. These genes also express protein isoforms lacking the second half of the RRM (FoxDeltaRRM), due to the skipping of a highly conserved 93-nt exon. Fox binding elements overlap the splice sites of these exons in Fox-1 and Fox-2, and the Fox proteins themselves inhibit exon inclusion. Unlike other cases of splicing autoregulation by RNA-binding proteins, skipping the RRM exon creates an in-frame deletion in the mRNA to produce a stable protein. These FoxDeltaRRM isoforms expressed from cDNA exhibit highly reduced binding to RNA in vivo. However, we show that they can act as repressors of Fox-dependent splicing, presumably by competing with full-length Fox isoforms for interaction with other splicing factors. Interestingly, the Drosophila Fox homolog contains a nearly identical exon in its RRM domain that also has flanking Fox-binding sites. Thus, rather than autoregulation of splicing controlling the abundance of the regulator, the Fox proteins use a highly conserved mechanism of splicing autoregulation to control production of a dominant negative isoform.
Figures
FIGURE 1.
Diagram of the mouse Fox genes. (A) The exons are shown as boxes. The coding sequence is shown in light gray, and the alternative in-frame start codons are indicated. The dark-gray box represents the alternative exon encoding the second half of the RRM domain. The exon annotation of Fox-1 and Fox-2 is as described for the human genes (Underwood et al. 2005). Exons 1C.1 and 1D.1 of Fox-1 derive from mouse-specific promoters. Alternative exons B40, M43, and A53 (Nakahata and Kawamoto 2005) and the FAPY C-terminal splice isoforms are also indicated. Fox-2 exon 13 (dashed box) is seen in human but not found in mice. The tissue specificity of the mouse Fox first exons is indicated below. The relative expression of these exons in mouse brain, heart, and skeletal muscle are compared in Supplemental Figure S1. Alignment of human and mouse alternative Fox RRM exons, the corresponding chick, frog, fish, and fly Fox exons, and the flanking splice sites (B). The sequences are grouped according to their similarity to the human Fox genes. The corresponding protein coding regions of the worm Fox genes are shown below. The exon and the UGCAUG elements are marked by open and gray boxes as indicated.
FIGURE 2.
Fox alternative RRM exon splicing in adult mouse. (A) The inclusion/skipping of Fox-1 exon 11, Fox-2 exon 6, and Fox-3 exon 8 in adult cerebellum, cortex, striatum, heart, and muscle was studied by RT-PCR. The bands marked by an asterisk represent incompletely denatured PCR product. Fox-2 exon 6 skipping is induced in differentiating C2C12. (B) Mouse myoblastoma C2C12 were stimulated to form myotubes in medium containing 2% horse serum. Cells were harvested 0, 2, 4, and 6 d after starting the differentiation. The splicing of Fox-2 exon 6 was studied by RT-PCR. Analysis of the muscle Fox proteins by Western blotting. (C) Protein lysate from purified mouse skeletal muscle nuclei were probed by antibodies specifically recognizing all Fox proteins with an intact RRM (lane αFoxRRM; see also Fig. 5B), Fox-1-specific antibody (lane αFox-1), and anti-Fox-2 antibody (lane αFox-2). The Fox-2ΔE6 isoforms (right) are detected only by the anti-Fox-2, but not by the anti-RRM or the anti-Fox-1 antibodies.
FIGURE 3.
The alternative RRM exon encodes the second half of the Fox RRM. (A) The RRM domain of mouse Fox-2 is aligned with the RRM model SMART00360 by a search engine for conserved protein domains (
http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
). The characteristic RNP1 and RNP2 features are boxed and the secondary structure is drawn to scale below. Skipping of the alternative exon causes deletion of the second half of the RRM, including β-strand 3 and α-helix 2. Fox-2ΔE6 binds RNA weakly in vivo. (B) FLAG-tagged Fox-2 and Fox-2ΔE6, lacking exon 6, were transiently expressed in HEK293 cells and UV-cross-linked in vivo. The FLAG-tagged proteins were affinity purified from cellular lysates pretreated with RNase A. The RNA–protein cross-links were 32P-labeled. After separation by SDS-PAGE, the proteins were transferred to PVDF membrane. (Left) The RNA–protein cross-links were detected and quantified by PhosphorImager and the total protein quantified by FLAG Western. (Right) The normalized RNA binding of Fox-2 and RBMΔE6.
FIGURE 4.
Fox-1 and Fox-3, but not their ΔRRM isoforms inhibit the inclusion of Fox-2 exon 6. (A) RT-PCR analysis of the endogenous Fox-2 exon 6 splicing. Human HEK293 and mouse neuroblastoma N2A cells were transiently transfected with FLAG-tagged Fox-1, Fox-1ΔE11, Fox-3, and Fox-3ΔE8 as indicated. (Lanes −) A control transfection with vector not bearing a protein reading frame was carried out in parallel. (Lanes −/−) Samples prepared similarly to the adjacent lanes −, except that reverse transcriptase was omitted. The fraction of Fox-2 mRNA excluding exon 6 is shown below each lane as a percent of the total. (B) The recombinant proteins were detected by Western blotting using anti-FLAG and anti-FoxRRM antibodies. Anti-U1 70K Western is carried out in parallel as a loading control.
FIGURE 5.
Fox-2ΔE6 inhibits Fox-dependent activation of splicing. (A) HEK293 cells were transfected with the minigene DUP-E33CACNA1Cwt, containing a Fox-dependent alternative middle exon. A constant amount of FLAG-Fox-2 was cotransfected with increasing amounts of FLAG-Fox-2ΔE6. (Lane −) A control transfection with the minigene only was performed in parallel. (Top) The inclusion of the alternative exon was determined by RT-PCR. The percent exon inclusion is given below. (Middle) The amounts of FLAG-Fox-2 and FLAG-Fox-2ΔE6 were measured by anti-FLAG western. (Bottom) Anti-U1 70K Western served as a loading control. The relative FLAG-Fox-2 levels, shown below each lane are normalized to the U1-70K content. The molar ratios of Fox-2 to Fox-2ΔE6 are indicated. The effect of Fox-2ΔE6 on Fox-1-dependent and Fox-3-dependent splicing of the DUP-E33CACNA1Cwt minigene was studied in a similar way and is shown in B and C, respectively.
FIGURE 6.
Fox-1ΔE11 and Fox-3ΔE8 inhibit Fox-dependent activation of splicing. (A) The minigene DUP-E33CACNA1Cwt, FLAG-Fox-1, and increasing amounts of FLAG Fox-1ΔE11 were cotransfected in HEK293 cells. (B) A similar experiment was carried out with Fox-3 titered with Fox-3ΔE8. (Lanes −) Control transfections with the minigene only were performed at the same time. Lanes −/− were prepared similarly to lanes −, except that reverse transcriptase was omitted. (Top) The splicing of the minigene was analyzed by RT-PCR. The percent of exon inclusion is given below. (Middle) The amounts of Fox and FoxΔRRM were measured by anti-FLAG western. Anti-U1 70K Western served as a loading control. The relative FLAG-Fox levels, shown below each lane are normalized to the U1-70K content. The molar ratios of Fox to Fox-2ΔRRM are also indicated. (Bottom) The effect on splicing of exon 6 of the endogenous of Fox-2 was tested by RT-PCR. The fraction of the Fox-2 mRNA skipping exon 6 is shown below each lane.
FIGURE 7.
Fox-2ΔE6 does not alter Fox-dependent splicing repression. HEK293 cells were transfected with the minigene DUP-E9*CACNA1Cwt, containing a middle exon inhibited by Fox. FLAG-Fox-2 and FLAG-Fox-2ΔE6 were cotransfected into these cells as in Figures 5 and 6. The splicing of the middle exon and the expression of the Fox proteins were determined as above.
FIGURE 8.
Diagram of the regulation of Fox expression and activity through splicing of the alternative RRM exon. Fox pre-mRNA, mRNA, and protein isoforms are shown from left to right. The alternative exon, encoding the second half of the RRM motif is shown as a gray box, its flanking constitutive exons as open boxes. The blunt arrows indicate an inhibitory effect.
References
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