Role of Polyadenylation Site in Post-Transcriptional Regulation of the Thyroid Hormone Receptor Gene

Angela Schnell
Marquette University
Milwaukee, WI
Mentor: Dr. Stephen Munroe

Within the human genome, and those of other mammals, far fewer protein coding genes have been found than originally predicted.  Instead complexity results from genes that give rise to multiply spliced and polyadenylated transcripts.  This project focuses on the regulation of alternatively processed mRNAs and the relationship between termination, splicing and polyadenylation.  Our lab has studied the alpha-thyroid-hormone receptor (TR?) gene as a model system to understand alternative processing.  TR?, in mammals, produces two mRNAs that are identical except for their 3’ exons: TR?1 and TR?2.  The mRNAs have separate poly(A) sites with the TR?1 site lying upstream of the TR?2 site.

To characterize the process by which the two mRNAs are alternatively processed and selection between them is made, mutational analysis on the poly(A) sites for TR?1 and TR?2 was performed.  Previous work has shown that the TR?1 poly(A) site and 5’ TR?2 specific splice site played competing roles in selection between the two transcripts.  Constructs replacing the both the TR?1 and TR?2 poly(A) sites with the SV40 poly(A) site, a well characterized stronger poly(A) site, were made.  293 cells were transfected with these constructs and several related previously made constructs.  The RNA was collected and RNase protection assays were performed to determine the relative mRNA levels of TR?1 and TR?2.

My results replicated previous results that the TR?1 poly(A) site and TR?2 specific 5’ splice site were important signals for the decision between the two transcripts.  Furthermore, we found that the strong SVL poly(A) site at the TR?2 site also alters mRNA levels enhancing the amount of TR?2.  Thus, it is likely that transcription extends beyond the TR?2 poly(A) site before selection between the two transcripts is made.  This suggests at least two possible mechanisms.  Either polyadenylation at the downstream site commits it to becoming the pre-mRNA to become TR?2 or the strong downstream poly(A) site may enhance splicing at the TR?2 specific splice sites through a process called “exon definition.”  Additional experiments will be performed to characterize the TR?2 poly(A) site’s effect demonstrated in my summer research. 
 
 
 

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