The Tgif2 gene contains a retained intron within the coding sequence

Abstract

Background: TGIF and TGIF2 are homeodomain proteins, which act as TGFbeta specific Smad transcriptional corepressors. TGIF recruits general repressors including mSin3 and CtBP. The related TGIF2 protein functions in a similar manner, but does not bind CtBP. In addition to repressing TGFbeta activated gene expression, TGIF and TGIF2 repress gene expression by binding directly to DNA. TGIF and TGIF2 share two major blocks of similarity, encompassing the homeodomain, and a conserved carboxyl terminal repression domain. Here we characterize two splice variants of the Tgif2 gene from mouse and demonstrate that the Tgif2 gene contains a retained intron.

Results: By PCR from mouse cDNA, we identified two alternate splice forms of the Tgif2 gene. One splice variant encodes the full length 237 amino acid Tgif2, whereas the shorter form results in the removal of 39 codons from the centre of the coding region. The generation of this alternate splice form occurs with the mouse RNA, but not the human, and both splice forms are present in all mouse tissues analyzed. Human and mouse Tgif2 coding sequences contain a retained intron, which in mouse Tgif2 is removed by splicing from around 25-50% of RNAs, as assessed by RT-PCR. This splicing event is dependent on sequences within the mouse Tgif2 coding sequence. Both splice forms of mouse Tgif2 encode proteins which are active transcriptional repressors, and can repress both TGFbeta dependent and independent transcription. In addition, we show that human and mouse Tgif2 interact with the transcriptional corepressor mSin3.

Conclusion: These data demonstrate that the Tgif2 gene contains a retained intron, within the second coding exon. This retained intron is not removed from the human mRNA at a detectable level, but is spliced out in a significant proportion of mouse RNAs. This alternate splicing is dependent entirely on sequences within the mouse Tgif2 coding sequence, suggesting the presence of an exonic splicing enhancer. Both splice forms of mouse Tgif2 produce proteins which are functional transcriptional repressors.

Figure 1

Two splice forms of mouse Tgif2. A) Expression of mouse Tgif2 was analyzed by PCR from 1^st strand cDNA, using primers at the 5' and 3' ends of the coding sequence. cDNAs were from the tissues indicated (sk. musc = skeletal muscle), or from embryos at the indicated days of gestation. -ve: no cDNA. Similar PCR analysis was carried out on cDNA from human brain and kidney (right). B) Alignment of putative splice sequences from mouse and human TGIF2 with splice consensus sequences (matches to the consensus are shaded gray).

Figure 2

Multiple forms of the mouse TGIF2 protein. A) alignment of human TGIF2 and mouse Tgif2 and Tgif2d protein sequence. Differences between human and mouse are shaded. Dashes indicate amino acids missing in mTGIF2d. The homeodomain and carboxyl-terminal extension are boxed. The carboxyl-terminal region of TGIF2 which is conserved in TGIF is indicated by a dashed box. MAP kinase phosphorylation sites are boxed, and threonine to valine mutations are indicated below. B) Alignment of the carboxyl-terminal part of the homeodomain and extension from TGIFs of different species. TGIF2: human, mouse; TGIF: human, mouse, chicken, Xenopus, zebra fish; Drosophila dTGIFa and dTGIFb. Residues identical between all are shaded black, similar are gray. The homeodomain is boxed and the arrow indicates the position affected by alternate splicing of mTgif2. COS-1 cells were transfected with the indicated Flag-tagged expression constructs (C) or with Flag-mTGIF2 or a mutant in which two threonines were altered to valine (D). Protein expression was analyzed by western blotting with a Flag specific antibody.

Figure 3

The mouse Tgif2d protein is a functional repressor. A) COS-1 cells were cotransfected with myc-tagged mSin3A and the indicated Flag-tagged expression constructs. Proteins were isolated on Flag agarose and analyzed for coprecipitating mSin3A with a myc antibody. Expression of transfected proteins in cell lysates is shown below. B) HepG2 cells were transfected with a reporter in which the luciferase gene is driven by the TK promoter and two TGIF binding sites, together with increasing amounts of mTgif2 or mTgif2d. Luciferase activity was assayed after 36 hours and is shown compared to a control transfection (mean +/- s.d. of triplicate transfections). C) COS-1 cells were cotransfected with a Smad2 or Smad3 expression vector and the indicated Flag-tagged expression constructs. Proteins were isolated on Flag agarose and analyzed for coprecipitating Smad2 or Smad3. Expression of transfected proteins in cell lysates is shown below. D) HepG2 cells were transfected with the 3TP-lux reporter, together with human TGIF, TGIF2 or mouse Tgif2d. After 24 hours, cells were treated with TGFβ (100 pM), or left untreated and luciferase activity was assayed 16 hours later, and is shown as the mean +/- s.d. of triplicate transfections.

Figure 4

A retained intron in the TGIF2 gene. A) RT-PCR analysis of COS-1 cells transfected with mouse or human TGIF2 (lanes 1 and 2) or direct PCR on plasmid DNA (lanes 3 and 4). Positions of the PCR primers are shown to the right. B) A schematic representation of splicing events of the mouse Tgif2 RNA is shown.

Figure 5

The 3' coding region of mouse Tgif2 is required for alternate splicing. COS-1 cells were transfected with expression constructs and expression of human, mouse and chimeric TGIF2 RNAs was analyzed by RT-PCR. The positions of primers are indicated (arrows). Alternate splicing: + splicing, - no splicing, -/+ just detectable, +/- slightly reduced. Constructs are shown schematically: Gray = mouse, black = retained intron. Stripes = human, crosshatch = retained intron. A) Comparison of constructs in which regions 5' and 3' of the retained intron are swapped between human and mouse TGIF2. B) Comparison of splicing of 3' deletion constructs for human and mouse TGIF2. C) Analysis of a series of 3' deletions of mouse TGIF2.

Figure 6

The branch site sequence contributes to splicing efficiency. A) Chimeric forms of Tgif2 were analyzed for splicing of the retained intron. A mutation of the branch site was introduced into the human TGIF2 sequence. HhH: the sequence of the branch acceptor site in the human was altered to that of mouse, HhM: as HhH, with mouse sequence 3' to the retained intron. HMh and MHh: the region 3' of the retained intron consisted of human sequences to base 543 and mouse sequence thereafter. The other regions of the coding sequence were human (H) or mouse (M) as indicated. B) Comparison of the sequences of the splice sites surrounding the retained intron and the branch acceptor site. Sequences from mouse (m), human (h), rat (r), dog (d) and cow (c) are shown with the consensus above. Boxed bases differ from the consensus. C) Pairwise sequence comparison of the coding sequence 3' of the retained intron (upper) or the entire coding sequences (lower) between the five species indicated. Percent identity is shown.