The Study On Newly Discovered Thyroid Hormone Receptor ? Isoforms

Thyroid hormone receptors (TRs), as the member of nuclear receptor superfamily, are coded by two genes,TRa and TRB. Because of different transcript start position and alternative splicing, each subtype has several isoforms, including TRal, TRa2, TRa3, TRB1, TRB2, TR?3and truncated TRAal, TRAa2, TR??3. Recently a new isoform of thyroid hormone receptor beta (TR?) in rat liver was found by us in our laboratory; it is not another truncated TR beta subtypes (for example TRA?3), but a new extended subtype with a long DNA-binding domain, named TR B A (not TR AB). This paper continue to study its origin, generating process and several tissue-distributions; At the same time we were lucky to discover another new isoform named TR?2?. This paper will be divided into three parts:TR?A generated by alternative splicing at protein level; The research of one more novel TR B isoform(TR?2?);The preliminary exploration of other membes in the nuclear receptor superfamily members DBD whether there is a similar new exon unknown.In order to study the existence of TR??splicing at the protein level, we construct TRPA minigene, including 3'section of exon3, whole length intron3-4, and 5' section of exon4, followed by reporters fluorescent protein or luciferase. We take the minigene for the transcription and the splicing experiments earring out in different cell lines; Enhanced green fluorescent protein or luciferase reporter genes were respectively fused at the 3'terminus of minigene for facilitate detection. Minigenes mutated by site-directed mutagenesis were used for experiments of splicing and translation in the absent or present of T3. RT-PCR assays showed:TRP A minigene have been successfully transcribed and spliced into two transcripts; At protein level: (1) Green fluorescence formation and luciferase activity value in the spliced exon 3/N/4 formation were significantly higher than those of exon 3/4; (2) The value of luciferase activity in T3 treatment group is significantly higher than the group without T3.Therefore, TRP A minigene are spliced into exon 3/N/4 in terms of mRNA or protein levels, and the spliced products is positive regulated by its ligand.To further study the TR??expression patterns in different tissues, we analyzed the content of TR??, TR?1 and the sum of TR??+TR?1 transcripts in the adult rat liver,brain, heart, lung, kidney, spleen,skeletal muscle and testicular using real-time quantitative PCR methods. The results showed that:TR??and TR?1 exwere pressed in all above-mentioned tissues except testicular,and it is highest in heart, low in brain.The TR??expression in each tissue was much higher than TR?1, and has a very large proportion in the total sum of TR??+TR?1.One more novel TR?isoform is initially discovered in the rat pituitary by us and the sequence submitted to Genbank was named TRbeta2Delta(TRb2?HM043807.1). TR?2?, TR?2 and TR?2?+TR?2mRNA in the adult rat pituitary tissue distribution were analyzed with real-time quantitative PCR, the results showed the TR?2 and the TR?2?mRNAs in pituitary tissue are almost in the same amount. The newly discovered TR?2?cDNA coding sequence was cloned into fusion prokaryotic expression vector pQE-30Xa and expressed in E.coli. The recombinant protein was identified with SDS-PAGE and western-blotting.TR?2?fusion protein reached 20.21mg/L medium. The interesting protein accounted for 26.3%of the total bacterial protein. The recombinant protein containing 6×His tag was purified with Ni-NTA resin affinity chromatography and used to electrophoretic mobility shift assay (EMSA) and radioligand binding assay (RLBA). The results indicated that recombinant TR?2?can both bind with the target DNA and the T3 specifically. We constructed pcDNA3.1/TR?2?eukaryotic expression vector and PAL TRE vector containing the pGL3-Promoter preceding the reporter gene,and they were co-transfected COS-7 cells. The results show:TR?2?binding its ligand and TRE to promote transcription of target genes, further evidence confirms that the TR?2?is a new subtype of functional TR?.We chose four membes in the nuclear receptor superfamily members for researching their variant. Specific amplification primers designed to detect the existence of the new unknown exons in their DBD. We have failed to find another DBD encoded by one more exon like TR??.