In Vitro Protein Level Study Of A Novel Splicing Pattern In Thyroid Hormone Receptor β Pre-mRNA

Thyroid hormone receptor (TR), as the member of nuclear receptor superfamily, is expressed in almost all tissues of the body. Thyroid hormone plays an important role in body's metabolic and developmental regulation through binding to TR and exerting most of their functions at the genomic level. There are two different genes that encode two different TRs, TR alpha and TR beta. Because of different transcript start position and alternative splicing, each subtype has several isoforms, including TRα1, TRα2, TRα3, TRΔα1, TRΔα2, TRβ1, TRβ2, TRβ3, TRΔβ3. TRβΔis a novel TRβisoform which was isolated and identified in rat liver. We found a new exonN between exon3 and exon4. In this paper, a study of two ways of splicing in rat liver cell is reported.Construction of recombinant plasmid:The target fragment Luciferase, EGFP, cDNA 3N4, cDNA34 and Minigene were obtained by PCR. The recombinant plasmid pcDNA3.1(+)/Luciferase,pcDNA3.1(+)/EGFP,pcDNA 3.1(+)/cDNA 3N4-Luciferase,pcDNA3.1(+)/cDNA 3N4-EGFP,pcDNA3.1(+)/cDNA34-Luciferase,pcDNA3.1(+)/cDNA34-EGFP,pcDNA3.1(+)/Minigene-Luciferase and pcDNA3.1(+) /Minigene-EGFP were constructed by restriction site. Sequence analysis and restriction enzyme digestion confirmed successful construction of the recombinant expression vector.Mutagenesis of recombinant plasmid:One base of exonN in plasmid pcDNA3.1(+)/Minigene-Luciferase and pcDNA3.1(+)/Minigene-EGFP was deleted by in vitro site-directed mutagenesis techniques, constructing recombinant plasmid pcDNA3.1(+)/Minigene-Ndel1-Luciferase, pcDNA3.1(+)/Minigene-Ndel1-EGFP. Two bases of exon4 in plasmid pcDNA3.1(+)/Minigene-Ndel1-Luciferase, pcDNA3.1(+)/Minigene-Ndel1-EGFP were deleted by in vitro site-directed mutagenesis techniques, constructing recombinant plasmid pcDNA3.1(+)/Minigene-Ndel1-4del2-Luciferase, pcDNA3.1(+)/Minigene-Ndel1-4del2-EGFP. Sequence analysis confirmed successful mutagenesis of the recombinant expression vector.In vitro transfection:The amount of the plasmids for transfection was adjusted to the same mole and transfected into rat liver cell BRL.48 hours later, the results were analyzed by RT-PCR, inverted microscope and Luciferase Assay. Two bands were identified in RT-PCR, indicating that alternative splicing produced two isoforms. Observations of green fluorescence showed that green fluorescence was clearly visible in the cells transfected with plasmid pcDNA3.1(+)/Minigene-EGFP, green fluorescence was nearly invisible in the cells transfected with plasmid pcDNA3.1(+)/ Minigene-Ndel1-EGFP, and green fluorescence was clearly visible in the cells transfected with plasmid pcDNA3.1(+)/Minigene-Ndel1-4del2-EGFP. Analysis of Luciferase activity showed that the difference between the cells transfected with plasmid pcDNA3.1(+)/Minigene-Luciferase and pcDNA3.1(+)/Minigene-Ndel1-Luciferase was statistically significant (p<0.05), the difference between the cells transfected with plasmid pcDNA3.1(+)/Minigene-Ndel1-4del2-Luciferase and pcDNA 3.1(+)lMinigene-Ndel-Luciferase was statistically significant (p<0.05), and the difference between the cells transfected with plasmid pcDNA3.1(+)/Mingene-Luciferase and pcDNA3.1(+)/Minigene-Ndel1-4del2-EGFP was not statistically significant (p>0.05).In summary, above results revealed that TRβΔminigene model can be spliced into two transcripts in rat liver cells. And we can believe that TRβΔminigene recombinant plasmid was mainly spliced into exon 3N4 in rat liver cells.