Determination of Yeast TRAMP Complex Conservation in Mouse

Andrew Bestul
Marquette University
Milwaukee, WI
Mentor: Dr. Stephen Munroe

The TRAMP complex is a unique nuclear surveillance pathway that regulates expression and processing of various RNAs in yeast before export to the cytoplasm. This complex is composed of three proteins: Trf 4/5p, Air 1/2p and Mtr4p. The Trf4 protein has been identified as a polyadenylation polymerase that polyadenylates the targeted RNA for this specific pathway (Kadaba et al. 2004). The Air 1/2 proteins are zinc finger proteins that are potential RNA-binding subunits. Mtr4 proteins are RNA helicases. The target RNA for this pathway includes pre rRNAs, pre-tRNAs and even non-coding mRNAs. The polyadenylated RNA is then sent to the nuclear exosome. In the exosome, the target RNA is recognized by the exonuclease, Rrp6p and degraded.

The proteins in this complex are conserved in the mouse genome. There are homologs to each of the yeast proteins. The experiment is to see how knockdown of these TRAMP genes in mouse will affect its phenotype. When components of the TRAMP pathway were deleted in yeast, a large increase in certain polyadenylated RNAs was seen. There were also amounts of previously unseen non-coding mRNA in the mutant yeast cells (Wyers et al. 2005). This is what might be expected in mouse cells if TRAMP complex function is conserved. 

We will assay for the function of mammalian homologs of TRAMP proteins by knocking them down and identifying changes in the amounts of polyadenylated RNA. Also, we are interested in determining whether new RNAs are seen in these knockdowns compared to normal cells. To knockdown the homologous mouse genes, two methods will be used to introduce siRNA. The first method, mouse neuro 2A cells (ATTC # CCL-131) are directly transfected with synthetic 21-nt siRNAs targeted against the mouse Mtr4 gene, SKIV2L2 (GenBank #: NM_028151). The second method involves a lentiviral system. This system will combine different viral proteins with the main transfer vector, the LentiLox 3.7. Several different 60 bp inserts have been cloned into a derivative of LentiLox 3.7 so that they are transcribed in vivo by the mouse cells to yield small hairpin RNAs (shRNAs). Recombinant viruses expressing these shRNA inserts will be used to infect neuro 2A cells and knockdown the mouse Trf4/5 homologous gene, Trf 4-2 (GenBank #: XM_134422).
 
 
 
 

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