- Research article
- Open Access
Unusual misregulation of RNA splicing caused by insertion of a transposable element into the T (Brachyury) locus
© Goldin and Papaioannou; licensee BioMed Central Ltd. 2003
- Received: 3 December 2002
- Accepted: 17 April 2003
- Published: 17 April 2003
The T Wis mutant allele of the Brachyury, or T, gene was created by insertion of an endogenous retrovirus-like early transposon (ETn) element into the exon 7 splice donor consensus sequence of the 8 exon T locus. While the developmental consequences of this disruption have been well characterized, the molecular consequences have not been previously investigated, and it has been assumed that the insertion results in a truncated protein. This study sought to further characterize the mutant T Wis allele by investigating the nature of the transcripts produced by insertion of this transposable element.
Using an RT-PCR based approach, we have shown that at least 8 different mutant transcripts are produced from the T Wis allele. All T Wis transcripts bypass the mutated exon 7 splice donor site, such that wild type T transcripts are not produced from the T Wis allele.
This result shows an unsuspected misregulation of RNA splicing caused by insertion of a transposable element, that could have more widespread consequences in the genome.
- Mutant Embryo
- Splice Donor Site
- Dominant Negative Effect
- Mutant Transcript
- Aberrant Transcript
The history of the T locus began with the discovery in 1927 of a semi-dominant mutation in mice, named Brachyury, or T for tail, that affects both embryonic viability in homozygotes and tail development in heterozygotes . This original T allele represents a deletion spanning 160–200 kb (reviewed in ), the developmental effects of which have been well characterized [3–8]. Homozygous mutant embryos show a developmental failure of the notochord and posterior mesoderm, and die at midgestation. Heterozygous mutant mice are born with shortened tails and malformed vertebrae. In 1988, another spontaneous Brachyury mutation, called T Wis , was reported . The T Wis homozygous and heterozygous mutant phenotypes are more severe than those of the T deletion, suggesting that the T Wis allele acts as a dominant negative. Homozygous mutants have no somites at all and heterozygotes have no tail, rather than a shortened tail (reviewed in ).
The T gene was cloned in 1990 , and its expression pattern was found to correlate with the tissue types affected in T mutants . Subsequently, T protein was shown to bind specifically to DNA and its preferred in vitro target sequence was identified . It was further shown that T encodes a transcription factor capable of regulating expression of a reporter via the identified target sequence . In their report describing the initial cloning of the T gene, Herrmann et al.  also demonstrated that the T Wis allele of T results from the insertion of an endogenous retrovirus-like early transposon (ETn) element into the splice donor site of exon 7 of the 8 exon T locus. They showed that the splice site at the 3' end of exon 7 was mutated from TAG GTATGT to TAG GTGTTG (where underlined sequence is the 3' end of exon 7 and ETn insertion sequence is in bold) and predicted that this site would be nonfunctional, thereby abolishing splicing of exon 7 to exon 8. They proposed that the T Wis allele would produce a transcript comprised of exons 1 through 7 followed by read-through transcription of the ETn element, resulting in a modified C-terminal end, or alternatively, that upstream splice donor sites would be used, shortening the transcript and protein product. In later whole mount in situ analysis, Herrmann  confirmed expression of a T locus-derived transcript in T Wis /T Wis embryos, but did not examine its exact nature. The position of the ETn insertion (see Figure 3A) is compatible with the idea that a modified protein could still bind DNA but might compete with the wild type protein or form an inactive complex with it, thus leading to a dominant negative effect.
The aim of this study was to characterize the sequence and structure of the T Wis allele transcript(s), with an eye towards understanding the specific molecular consequences of disruption of the exon 7 splice donor site by the inserted ETn element. Using an RT-PCR-based approach, we have shown that at least 8 different transcripts are produced from the T Wis allele of T. In addition to the mutant transcript predicted by Herrmann et al. , 4 transcripts composed solely of T exonic sequences, and 3 transcripts containing ETn sequences spliced between T exonic sequences were identified. All T Wis transcripts bypass the mutated exon 7 splice donor site, such that wild type T transcripts cannot be produced from the T Wis allele.
Identity of the T Wis ETn element
Transcripts produced from the T Wis allele of T
Primers pairs used for RT-PCR analysis of T versusT Wis transcripts. See Materials and Methods for primer sequences.
Product Size (in bp)
T exon 6
T exon 8
T exon 4
T exon 8
T exon 6
T exon 7, last 35 bp
T exon 8
Sequence analysis of RT-PCR products fromT +/+,T Wis /+, and T Wis /T Wis embryos. RT-PCR products are named as described in Figures 2 and 3. Products of indicated size were sequenced and compared toT mRNA sequence and genomic structure (Herrmann, Labeit et al. 1990; Genbank accession number NM-009309 ). Transcript sequence indicates exonic organization of RT-PCR products.
Product Size (in bp)
6,7,8 (wild type)
T Wis /T Wis
6,7Δ35,8 & 6,7Δ35,+37ETn,8
T Wis /T Wis
4,5,6,7,8 (wild type)
T Wis /T Wis
4,5,6,7Δ35,8 & 4,5,6,7Δ35,+37ETn,8
T Wis /T Wis
T Wis /T Wiss
T Wis /T Wis
7,8 (wild type)
T Wis /T Wis
7+60Etn,8 & 7+60ETn,+37ETn,8
As expected, the F1-BGH037 primer set failed to amplify from T +/+ embryo RNA (Figure 1B). In the T Wis /T Wis embryo, this primer set detected a mutant transcript representing read-through transcription from exon 7 into the immediately 3' ETn insertion (6,7+Etn; Figure 1B, Table 2, Figure 3C).
The characterization of T Wis allele transcripts presented here confirms that the intron 7 donor splice site is indeed non-functional such that wild type T mRNA is never produced. Furthermore, the exon 1–7+ETn transcript predicted by Herrmann et al.  was identified. However, at least 7 other T Wis mutant transcripts are also produced (Figure 3C). All of these transcripts serve to bypass the mutated splice donor site; either by splicing over exon 7, activating a cryptic splice site within exon 7, or transcribing through exon 7 into the adjacent ETn sequences. The cryptic exon 7 splice site used, CCT GTGAGT, is a strong match to the C/A,AG GT,A/G,AGT splice donor consensus. It is important to note that in addition to the exon 4 to 8, exon 5 to 8, and exon 6 to 8 splicing events detected here, the potential remains that transcripts resulting from exon 1 to 8, exon 2 to 8, and/or exon 3 to 8 splicing events are produced but were not discovered.
Genetically, the T Wis allele acts as a dominant negative in that T Wis /T Wis embryos have a more severe phenotype than the null T/T embryos. However, the biochemical nature of this dominant negative effect is not known. This study confirms previous work  showing the existence of a transcript that would result in a truncated protein if translated. Because the DNA-binding T-domain would be intact, this hypothetical protein could compete with the wild type protein producing a dominant negative effect. Alternatively, the presence of heterologous Etn element sequences in T Wis transcripts may stimulate posttranscriptional gene silencing by an RNA-induced silencing complex (RISC)-based RNAi degradation pathway which could target degradation of wild type transcripts .
We have shown that disruption of a single splice donor site within a multi-exon locus can lead to a dramatic misregulation of RNA splicing. A straightforward prediction suggests that in T Wis transcripts, splicing "out of" exon 7 would simply fail and result in a transcript terminating with the ETn element. In reality the effect is much more complex, cautioning against the use of straightforward assumptions in predicting the molecular consequences of genetic alterations.
T Wis mice and embryo collection
The T Wis allele was maintained on a mixed A/J + C57BL/6 + 129/SvEv background. Embryos were collected from timed heterozygous matings. At e7.5 and e8.5, a portion of the extraembryonic region was removed for PCR genotyping, and the remainder of the embryos placed individually into 400 μL TRIZOL Reagent (Life Technologies, Inc.) for RNA preparation. At e9.5, mutant embryos were identified visually by absence of posterior embryonic tissues . For genomic PCR of the ETn insertion, yolk sac lysates from homozygous mutant e9.5 embryos were used. Genomic DNA from a wild type mouse served as a negative control. PCR was performed in 50 μL reactions containing: template, 50 pmol each of primers F3 (5'-CATAACGCCAGCCCACCTACTG-3') and ETn1 (5'-CACGATTTGTGGGTAAAATAGGAG C-3'), 2.5 units Taq DNA polymerase, 0.2 mM dNTPs, 20 mM TrisHCl (pH 8.4), 50 mM KCl, and 1.5 mM MgCl. Cycling parameters were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 67°C for 45 sec, and extension at 72°C for 90 sec, and a final extension at 75°C for 5 min. PCR products were electrophoresed through a 1.3% agarose TAE gel, excised from the gel, and purified using QIAEX II Gel Extraction Kit (Qiagen). Purified products were sequenced twice on each strand with the F3 and ETn1 primers using an ABI Prism Model 373 Automated DNA Sequencer. The ETn elements used to design the Etn1 primer were Genbank #s Y17106, Y17107, and X15598.
For genotyping of e7.5 and e8.5 embryos, PCR was performed on lysates of extraembryonic tissue as template, using primers BGH037 (5'-ACGTTGCGAGCTGCTGCGGC-3'), BGH039 (5'-ACCCATGTCAAACCCATCAG-3'), and BGH052 (5'-CCTATGCGGACAATTCATCTG-3'), in a similar reaction mix except for 1.0–1.5 mM MgCl. Cycling parameters were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 61° for 45 sec, and extension at 72°C for 45 sec, and a final extension at 75°C for 3 min. The primers BGH052 and BGH037 amplify an ~180 base pair (bp) fragment from the T Wis insertion allele, while primers BGH052 and BGH039 amplify an ~220 bp fragment from the wild type allele.
For RT-PCR, total RNA from individual e7.5 and e8.5 embryos was prepared essentially according to the TRIZOL Reagent manufacturer's protocols for isolation of RNA from small quantities of tissue. Total RNA was reverse transcribed using Superscript II RNase H- Reverse Transcriptase (Life Technologies, Inc.) as per manufacturer's protocol, except that all reactions were doubled to 40 μL final volume. RT reactions were performed on one half or one third of the total RNA isolated from an e7.5 or e8.5 embryo, respectively. For positive control reactions, RNA from one wild type embryo was used. Standard RT-PCR reactions were performed with each 50 μL reaction containing 1/8 of an RT reaction, 50 pmol each of forward and reverse primer, 2.5 units Taq DNA polymerase, 0.2 mM dNTPs, 20 mM TrisHCl (pH 8.4), 50 mM KCl, and 1.5 mM MgCl. Cycling parameters were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 65° or 64°C for 45 sec, and extension at 72°C for 45 sec, and a final extension at 75°C for 3 min. RT-PCR products were excised from gels, purified using the QIAEX II Gel Extraction Kit (Qiagen), and sequenced in an ABI Prism Model 373 Automated DNA Sequencer. Primers used for RT-PCR and/or sequencing were: F1 (5'-GTCATCGCCCTACCCCAG-3'), R1 (5'-GTGTGCGTCAGTGGTGTGTAATG-3'), F2 (5'-CCCATTTGCTAAAGCCTTCCTTG-3'), R2 (5'-AGGCACTCCGAGGCTAGACCAG-3'), BGH037 (5'-ACGTTGCGAGCTGCTGCGGC-3'), and F3 (5'-CATAACGCCAGCCCACCTACTG-3'). RT-PCR reactions using primers Hprt1a (5'-CCTGCTGGATTACATATAAGCACTG-3') and Hprt1b (5'-GTCAAGGGCATATCCAACAACAAAC-3') served as positive controls amplifying a 354 bp product from the ubiquitously expressed hprt transcript .
We wish to thank Timothy Bestor for encouragement and helpful discussion, Bernhard G. Herrmann for providing the genotyping protocols, sequence information and helpful discussion, and William Dove and A. Schedlovsky for providing the T Wis mice. This work was supported by grant RO1 HD33082 from the NIH.
- Dobrovolskaia-Zavadskaia N: Sur la mortification spontanée de la queue che la souris nouveau-née et sur l'existence d'un caractère (facteur) héréditaire "non viable". CR Seanc Soc Biol. 1927, 97: 114-116.Google Scholar
- Beddington RSP, Rashbass P, Wilson V: Brachyury – a gene affecting mouse gastrulation and early organogenesis. Develop. 1992, Supplement: 157-165.Google Scholar
- Gruneberg H: Genetical studies on the skeleton of the mouse XXIII. The development of brachyury and Anury. JEEM. 1958, 6: 424-443.Google Scholar
- Gluecksohn-Schonheimer S: The development of normal and homozygous Brachy (T/T) mouse embryos in the extraembryonic coelom of the chick. PNAS. 1944, 30: 134-140.View ArticleGoogle Scholar
- Rashbass P, Cooke LA, Herrmann BG, Beddington RSP: A cell autonomous function of Brachyury in T/T embryonic stem cell chimaeras. Nature. 1991, 353: 348-351. 10.1038/353348a0.View ArticlePubMedGoogle Scholar
- Herrmann BG: Action of the Brachyury gene in mouse embryogenesis. Ciba Foundation Symposium. 1992, 165: 78-91.PubMedGoogle Scholar
- Yanagisawa KO, Fujimoto H, Urushihara H: Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo. Develop Biol. 1981, 87: 242-248.View ArticlePubMedGoogle Scholar
- Wilkinson DG, Bhatt S, Herrmann BG: Expression pattern of the mouse T gene and its role in mesoderm formation. Nature. 1990, 343: 657-659. 10.1038/343657a0.View ArticlePubMedGoogle Scholar
- Shedlovsky A, King TR, Dove WF: Saturation mutagenesis of the murine t region including a lethal allele at the quaking locus. PNAS. 1988, 85: 180-184.PubMed CentralView ArticlePubMedGoogle Scholar
- Herrmann BG, Labiet S, Poustka A, King T, Lehrach H: Cloning of the T gene required in mesoderm formation in the mouse. Nature. 1990, 343: 617-622. 10.1038/343617a0.View ArticlePubMedGoogle Scholar
- Kispert A, Herrmann BG: The Brachyury gene encodes a novel DNA binding protein. EMBO J. 1993, 12: 3211-3220.PubMed CentralPubMedGoogle Scholar
- Kispert A, Koschorz B, Herrmann BG: The T protein encoded by Brachyury is a tissue-specific transcription factor. EMBO J. 1995, 14: 4763-4772.PubMed CentralPubMedGoogle Scholar
- Herrmann BG: Expression pattern of the Brachyury gene in whole-mount T Wis /T Wis mutant embryos. Development. 1991, 113: 913-917.PubMedGoogle Scholar
- Hammond SM, Caudy AA, Hannon GJ: Post-transcriptional gene silencing by double-stranded RNA. Nature Reviews Genetics. 2001, 2: 110-119. 10.1038/35052556.View ArticlePubMedGoogle Scholar
- Koopman P, Munsterberg A, Capel B, Vivian N, Lovell-Badge R: Expression of a candidate sex-determining gene during mouse testis differentiation. Nature. 1990, 348: 450-452. 10.1038/348450a0.View ArticlePubMedGoogle Scholar
- Clements D, Taylor HC, Herrmann BG, Stott D: Distinct regulatory control of the Brachyury gene in axial and non-axial mesoderm suggests separation of mesodermal lineages early in mouse gastrulation. Mechanisms of Development. 1996, 56: 139-149. 10.1016/0925-4773(96)00520-5.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.