The role of the Alu sequence of the TBXT gene in taillessness

A link between a single gene and a complex trait is very rare, but it makes headlines. There are about 140 genes associated with the absence of a tail. It was published in Nature that insertion of an Alu element (AluY) into intron 5 of the TBXT gene, which is paired in transcribed RNA with another inverted Alu element (AluSx1) in intron 6, results in alternative splicing of the Tbxt protein without exon 6. In mice, a similar change in this gene results in to loss of tail. The TBXT gene is involved in the development of the embryonic notochord, the precursor of the spine. At the end of the fourth week of human embryonic development, a tail appears, which disappears at the beginning of the eighth week, leaving a trace of the tail – the coccyx, where the spine ends. As a side effect, the Tbxt isoform lacking exon 6 causes neural tube defects in mice; something that occurs in one in a thousand human births. The loss of the tail may have contributed to upright walking, but it comes at the cost of an increased risk of neural tube defects.

I found it curious that it took so long to publish this article. His preprint It appeared on bioRxiv in September 2021 and generated widespread media attention. No one expected that its publication would take almost 900 days (2.5 years) after being sent to Nature. The reason is that their experiments with humanized mice were unsuccessful. The mutation observed in hominoids did not cause tail loss in all mice (it gave rise to the full spectrum of the trait, from tailless mice to mice with full tails). The first author, Bo Xia, who decided to study the topic for his doctoral dissertation at New York University (NYU) after a tailbone injury, needed a definitive, conclusive experiment. As stated in the paper itself, by pure chance he discovered that by inserting a 220 base pair sequence from intron 6 into intron 5 of the TBXT gene in mice, alternative splicing was achieved, similar to that in humans. With this idea he made the experiment a success; To do this, he inserted 297 base pairs (the same length as human AluY) from intron 5, but inverted, into intron 6; Thus, he simulated in mice the insertion observed in humans, and ensured that all mice were born without tails. This experiment confirmed his hypothesis and allowed him to pass peer review in Nature. In fact, Xia is already a postdoc at Harvard University and the Broad Institute (MIT/Harvard).

Of course, just because the TBXT gene has been discovered to play a role in taillessness does not mean that the taillessness problem has been solved. This is definitely a polygenic trait. There are at least 140 genes involved, and only one of them has been studied in detail. Future studies will need to clarify how all these genes interact with each other. Article by Bo Xia, Weimin Zhang, …, Itai Yanai, “On the genetic basis of the evolution of tail loss in humans and monkeys,” Nature 626: 1042-1048 (28 February 2024), doi: https://doi. org/10.1038/s41586-024-07095-8 (“Genetic basis for the evolution of tail loss in humans and apes,” BioRxiv preprint 460388, doi: https://doi.org/10.1101/2021.09.14.460388). For more information, see Miriam K. Konkel, Emily L. Casanova, “Mobile DNA sequence may explain tail loss in humans and monkeys,” Nature 626: 958-959 (28 February 2024), doi: https:// doi.org/10.1038/d41586-024-00309-z; and in Ewan Callaway’s intriguing book, “How Humans Lost Their Tails—And Why It Took 2.5 Years to Be Published,” News, Nature (February 28, 2024), doi: https://doi.org/10.1038/d41586 -024- 00610’s.

Non-hominoid primates have tails (except baboons, some macaques and lorises), unlike great apes (gibbons, orangutans, gorillas, chimpanzees and humans). This evolutionary divergence is thought to have occurred about 25 million years ago. Modern humans have 3 to 5 caudal vertebrae (variations among individuals) that give rise to the coccyx. To find the genetic reason for the absence of a tail in humans, the first step is to turn to bioinformatics tools. in the database Mouse Genome Informatics (MGI) discovered more than a hundred genes associated with the absence or shortening of the tail in mice. The most studied genes are Tbxt (Marriages), Wnt3a and Msgn1.

Bo Xia and his colleagues looked for genes in MGI that are marked by a “missing tail” (missing tail), 31 genes and with a “vestigial tail” (vestigial tail) or “short tail” (short tail), 109 genes. They then looked for orthologous genes for these 140 genes in primates. In eukaryotic cells like yours, genes are divided into introns (the non-coding part) and exons (the protein-coding part); The DNA of the gene is completely transcribed into pre-messenger RNA, which is processed by the splicing mechanism (splicewhich also translates as secure) to give rise to the final messenger RNA, which contains only exons. Said mRNA leaves the cell nucleus and is translated into proteins by ribosomes. Xia and his colleagues looked for differences in the exons of these 140 genes between various great and nonhominoid primates. They found 85,064 single nucleotide variants (SNVs), 5,533 deletions, and 13,280 hominoid-specific insertions. But none of them seemed promising for in-depth study.

For this reason, it was decided to look for non-coding sequences (introns) in these 140 genes. The Alu sequence in the sixth intron of the humanoid TBXT gene, called AluY, shone with its own light. This figure shows a multiple sequence alignment of the TBXT gene in 20 primates (with eight introns and eight exons, the latter in different colors). This gene contains two other Alu sequences, namely AluSx1 in the fifth intron, in the opposite direction to AluY, and AluSq2 in the seventh intron, in the same direction as AluY. By the way, sequences (sometimes called items) Alu are short pieces of DNA (about 300 base pairs) that behave like transposons (“mobile” DNA sequences that “jump” between generations from one piece of DNA to another). They are called Alu because they were discovered by the endonuclease Alu. It should be noted that these sequences are a genomic signature of primates (their evolutionary origins are estimated to date back to approximately 65 million years ago); mice lack Alu sequences.

The AluY sequence in the TBXT gene stands out because the senses of AluSx1 and AluY are opposite (as are the senses of AluSx1 and AluSq2). Moreover, the strands of AluSx1 and AluY are very similar, but point in different directions, so they can pair with each other in the secondary structure of the pre-mRNA. This suggests that they may be involved in an alternative splicing mechanism (alternative splicingor alternative splicing); In some eukaryotic genes, some exons are missing from the mRNA that were present in the pre-mRNA, meaning that a single gene in the DNA can produce several different proteins. To confirm this, Xia and colleagues used ViennaRNA software to reconstruct the secondary structure of mRNA. As can be seen from the figure, a long linear structure is observed due to the pairing of AluSx1 and AluY, which distances the sixth exon of the TBXT gene into the pre-mRNA of the fifth and seventh exons. Thus, when this pre-mRNA is processed to produce the final mRNA, the exon may be omitted. By the way, pairing may also exist between AluSq2 and AluSx1, leading to the formation of mRNA without the sixth and seventh exons; In this case, the embryonic development of the mice does not reach term, so it is not a viable option.

As a result of these alternative splicing mechanisms, transcription of the TBXT gene results in two different mRNAs: the complete one, with all exons one through eight, and the one associated with the loss of the humanoid tail, which lacks the sixth exon. To confirm this mechanism, studies were conducted on mutant mice for all of these variants. I won’t go into detail about these mouse studies (you know I’m structurally inclined). As I mentioned, the key point of these experiments is that the lack of Alu sequences in mice led us to model these sequences using an ingenious simulation experiment (an idea that will surely be used by other researchers interested in studying Alu sequences in model mice). The main finding of the mouse study is that there is a correlation between the relative abundance of the two Tbxt isoforms and the tail length of the mice.

Thus, it was shown that the insertion of the AluY sequence into the intron of the TBXT gene played an evolutionary role in the shortening or partial loss of the tail in the early ancestors of apes. However, it cannot be argued that this is the main cause of tail loss in hominoids. Additional genetic changes must have stabilized the tailless phenotype. For this reason, future research (from the 140 genes identified, or even new genes yet to be identified) is needed to identify the polygenic reason why you and I don’t have tails. Complex traits are always polygenic. But new research also takes us further. The human genome contains about 1.8 million copies of transposons, of which a million are Alu sequences (more than 60% are located in introns). A systematic search for pairs of opposite sense Alu sequences that could be associated with alternative splicing could help reveal their biological functions and possible impact on certain pathologies. As always in science, each new study is the first step on a long path that still lies ahead.