Epitope Tagging and Cellular Localization of Trf4p, a Novel Yeast Poly (A) Polymerase

Jacklyn Winters
Marquette University
Mentor:  Dr. James Anderson

The tRNA m1A58 (Mtase) is composed of two subunits encoded by two essential genes, TRM6 and TRM61.  A defective m1A58 Mtase leads to the destabilization of tRNAiMet.  This destabilization occurs through the coordinated action of Trf4p, a poly (A) polymerase and the nuclear exosome, a multiprotein complex with 3’?5’ exonuclease activity.  Our evidence suggests that m1A methylation occurs on nascent tRNA, either during or shortly after transcription is completed.  To determine cytologically if one component of the tRNA surveillance pathway, Trf4p, is discretely localized when actively polyadenylating tRNAiMet, we epitope tagged TRF4 in order to conduct indirect cellular immunofluorescence in wild type and trm6-504 mutant cells. 

TRF4 was epitope tagged with 3HA or 13 Myc in both wild type and trm6-504 mutant cells via PCR amplification, and yeast transformation.  The presence of the tags was verified through isolation of genomic DNA and Western blotting.  The 3HA epitope tagged Trf4p was expressed at very low levels, and exhibitied a phenotype similar to a TRF4 deletion, in a trm6-504 background, thus we concluded that Trf4Hap is not suitable for this study. Further investigation will be undertaken to N-terminus epitope tag the protein.  Should this tag allow proper Trf4p expression, immunofluorescence will be performed to localize the protein in the cell.
 

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