Antisense Overlap Between the TR?2 and Rev-erb? Nuclear Receptors is Truncated in Marsupials

Brandon Rindfleisch
Marquette University
Mentor: Dr. Stephen  Munroe

In mammals, the TR? gene codes for the alternatively spliced thyroid hormone receptor proteins TR?1 and TR?2, and overlaps with the antisense gene Rev-erb?. Rev-erb? is also part of the same nuclear receptor family as the TR? gene and is analogous to the TR?1 isoform. In eutherians (placental mammals), the TR?2 and Rev-erb? mRNAs have an overlap for 269 nucleotides with a bi-directional coding sequence that spans 201 nucleotides (Lazar, 1993). While TR?1 has a clear function and binds the thyroid hormone T3, TR?2 does not bind T3. TR?2 interferes with the other thyroid hormone receptors by lessening their transcriptional activation abilities in the presence of T3 (Lazar, 1993). TR?2 also is the major TR isoform expressed in various tissue types, such as the brain (Lazar, 1993). However, the actual physiological function of TR?2 remains unclear. 

Here we demonstrate characteristics of a divergent TR?2 in two marsupial genomes. One is Monodelphis domestica (Mikkelsen et al., 2007), also known as the gray short-tail opossum or the South American opossum.  The other is Didelphis virginiana or the North American opossum. A 1600 bp region of genomic DNA from D. virginiana, spanning a portion of the 3’UTR of the TR?1 gene to a portion of a downstream exon of Rev-erb?, was sequenced and compared to the recently published M. domestica. This analysis demonstrated that the TR?2 gene in both marsupials is truncated by more than 100 amino acids, its coding sequence stopping adjacent to the antisense stop codon of the Rev-erb? gene. Thus, instead of the bi-directional coding overlap in eutherians, the TR?2 and Rev-erb? coding sequences are non-overlapping in a tail-to-tail arrangement. However, at the C-terminal sequence of Rev-erb? the overlapping exon is completely conserved with respect to eutherians at the amino acid level, despite substitutions in the third position of many codons. 

In order to characterize expression of TR?2 mRNA, RNA will be obtained from various tissues of M. domestica and from two stages of development in collaboration with Dr. John VandeBerg at the Southwest Foundation for Biomedical Research.  RNA was reverse transcribed and amplified, revealing cDNA corresponding to the length expected for TR?2 cDNA. This product will be sequenced to confirm TR?2 mRNA expression.  The complete structure of the opossum TR?2 mRNA and its expression in different tissues relative to TR?1 and Rev-erb? mRNAs will be determined in subsequent experiments.  Hopefully, through the study of a TR?2 divergent from eutherians, the function of the variant TR?2 thyroid hormone receptor will be better understood.
 
 

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