Tra2beta as a novel mediator of vascular smooth muscle diversification

Abstract

Transformer splicing regulatory proteins determine the sexually dimorphic traits of Drosophila. The role of the vertebrate homologs of Tra-2 in phenotypic specification is undefined. We are using the alternative splicing of the MYPT1 E23 exon as a model for the study of smooth muscle diversification into fast and slow contractile phenotypes. Tra2beta mRNA and protein is expressed at up to 10-fold higher levels in fast smooth muscle tissues such as the rat portal vein and small mesenteric artery, in which E23 is spliced, as compared to the slow smooth muscle tissues of the large arteries and veins, in which E23 is skipped. Tra2beta is upregulated up to 10-fold concordant with the initiation of E23 splicing as the rat portal vein and avian gizzard implement the fast program of gene expression in the perinatal period. In disease models such as portal hypertension and mesenteric artery high/low flow, the portal vein and first order mesenteric artery dynamically downregulate Tra2beta concordant with a shift to E23 skipping and the slow program of gene expression. Tra2beta binds to a highly conserved sequence within E23 and transactivates its splicing in vitro and in vivo; this is abolished with mutation or deletion of this sequence. RNA interference-mediated knockdown of Tra2beta markedly reduces E23 splicing. We propose that Tra2beta has been conserved through evolution and redeployed for the specification of the fast smooth muscle phenotype and may serve as a novel nodal point for the investigation of this process in developmental and disease models.

Fig.1

MYPT1 3' E23 and flanking intronic sequence. A) Diagram of rat MYPT1 E23 sequence (gray box) and exon splicing. Phylogenetic sequence variation is underlined. Putative binding sites for splicing regulatory proteins are indicated above and below the exon. The splice site sequences are shown; nucleotides that deviate from the consensus are italicized. B) Phylogenetic analysis of rat MYPT1 genomic sequence consisting of E23 and flanking introns. Gray solid box,E23; line,flanking introns. The black histogram represents the evolutionary conservation among vertebrates. Horizontal lines, gaps in the alignment.

Fig.2

Concordance in Tra2β mRNA levels and MYPT1 E23 inclusion in smooth muscle. Tra2β mRNA abundance was measured by real-time PCR. The %E23 inclusion as measured previously is shown for reference purposes. (A) Tra2β increases 4-8 fold in the peri-natal period in the PV (upper panel) and Gz (lower panel) concordant with a complete switch to E23 inclusion (B) Tra2β is 5-10 fold more abundant in the mature PV, MA1 and Gz compared to large arteries and veins (Ao, IVC, PA), where E23 is nearly exclusively skipped. Cultured SMCs show low levels of Tra2β mRNA and skipping of E23. Values on the y-axis represent expression of Tra2β relative to A) and B) adult PV (upper panel) or Gz (lower panel), normalized to SRp20. Data is displayed as the mean ± S.D. (n ≥ 3). PV, Portal vein; Gz, gizzard; MA1, first order mesenteric artery; Aorta, Ao; IVC, inferior vena cava; PA, pulmonary artery; rasmc, rat aortic SMCs; GzSMC, gizzard SMCs; D, days post birth; ED, embryonic day, AD, adult.

Fig.3

Concordance in Tra2β mRNA levels and MYPT1 E23 inclusion in disease models. In A) PHT PV and B) HF and LF MA1 there is dynamic down-regulation in Tra2β mRNA concordant with a switch to skipping. Values on the y-axis represent expression of Tra2β relative to A) control PV and B) control MA1, normalized to SRp20. Data is displayed as the mean±S.D. (n ≥ 3). PHT, portal hypertension; HF, high-flow; LF, low-flow. D, Days after ligature of PV (PHT) or second order MA (MA1 HF/LF).

Fig.4

Tra2β isoforms in PV development and a disease model. A) Alternative splicing of exons 2 (black box) and 3 (striped box) gives rise to three major transcripts. The arrows indicate the positions of primers. The expected size of the PCR product (in bp) is indicated. Tra2β isoforms expression was analyzed in B) PV development and C) PHT PV. Representative gels are shown (n=3). bp, base pair.

Fig.5

Concordant up-regulation of Tra2β protein and MYPT1 E23 inclusion. Nuclear extracts from A) rat PV post-natal development and mature aorta and B) PV PHT were analyzed by Western blotting. Membranes were probed with antibodies to detect Tra2β and SR proteins. Approximate size of each band in kDa is indicated. A representative blot is shown (n=4).

Fig.6

Tra2β binds to the MYPT1 E23 in RNA pull-down assays. A) Sequence of wild type E23 and mutations and deletions of putative Tra2β and SR binding sites. B) The WT biotin-labeled RNA oligonucleotide was incubated with NE from adult rat PV, Ao, or D3 PHT PV. RNA-bound proteins were eluted and detected by western blotting. C) Oligonucleotides with deletion or mutation of Tra2β and/or SR binding sites were mixed with PV NE and analyzed as in B. The approximate size of the bound protein is indicated in kDa. A representative blot is shown (n=3). WT, wild type; Δ, deletions; mut, mutation; cont, control (unrelated RNA sequence).

Fig.7

Tra2β trans-activates MYPT1 E23 splicing dependent upon the exonic Tra2β cis-element. A) Diagram of MYPT1 mini-gene construct. Open box,βglobin exons; solid box, E23 exon; connecting lines,introns (thicker line,$\underset{.}{\beta }$ globin, thinner line,MYPT1). Arrows indicate the locations of the Primers. B) The wild-type MYPT1 mini-gene plasmid either alone (upper panel) or with a Tra2β expression plasmid (lower panel), was transfected into cultured chicken Gz or rat aortic SMCs, or injected into the gizzard in vivo. C) Dose response studies of Tra2$\underset{.}{\beta }$ expression plasmiḍ co-transfected with the wild type mini-gene into the RASMCs. A stoichiometry of MYPT1 minigene:Tra2β of 1:2 was optimum and was used in the other experiments. D) MYPT1 mini-gene mutation and deletion analysis. MYPT1 mini-gene constructs alone (upper panel) or with Tra2β expression plasmid (lower panel) were injected into the gizzard in vivo. RNA harvested 24 h post-transfection, and ratios of MYPT1 exon-in to exon-out mini-transcripts determined by RT-PCR. A picture of a representative gel is shown with quantification (n=3-6). *P<0.01 (WT+TRA vs. WT only), ^#P<0.01 (Δ/mut vs. WT)

Fig.8

Knock-down of Tra2β reduces MYPT1 E23 splicing. A) Tra2$\underset{.}{\beta }$ was knocked down in cultured HEK293 cells by siRNA. NEs were collected at 24h to 120h post transfection and Tra2$\underset{.}{\beta }$ level assessed by western blotting. Constant PTB levels are shown as internal controls. B) The wild-type MYPT1 mini-gene plasmid alone or with the Tra2β expression plasmid was transfected into 293 cells. The MYPT1 mini-gene plasmid was also transfected into 293 cells 48 h after treatment with siRNA against Tra2β, or GAPDH or ns siRNA as negative controls. RNA was isolated and ratios of MYPT1 exon-in to exon-out mini-gene transcripts were determined. Representative gels are shown with quantification as the mean ± S.D. (n≥3). *P<0.01 (vs. control), NS, non-specific.