Interactions Between the Ur Element RNA Oligonucleotide and CstF-64 in C. elegans Nematodes

In C. elegans, RNA processing of downstream operon genes through SL2 trans-splicing is crucial for polycistronic transcripts to be separated into monocistronic transcripts. The Ur element RNA sequence, which is important for proper SL2 trans-splicing, is found in the intercistronic region (ICR) of the pre-mRNA. The ICR is located between the 3’ end of one gene and the trans-splice site of a downstream operon gene. Lasda has shown that the Ur element sequence is required for downstream SL2 trans-splicing and proposed that it defines the 5’splice site on the SL2 RNA (Lasda, Allen, & Blumenthal, 2010). This project investigates whether there is an interaction between the Ur element RNA oligonucleotide and purified GST CstF-64, a 3’end formation factor. The Ur element RNA oligo contains short stem-loop and multiple UAYYUU motifs similar to Ur element RNA sequence found in ICRs. These experiments specifically analyze whether various mutant and wild type Ur element RNA oligos pull down GST CstF-64 directly, in the absence of extract. Results suggest that the wild type Ur element RNA oligo interacts directly with GST CstF-64, while the mutant RNA oligo does not pull down GST CstF-64. The results also suggest that the region containing the UAYYUU is required for GST CstF-64 binding, while the stem-loop is not important. Additionally, preliminary crosslinking results suggest that GST CstF-64 directly binds the loop and first UAYYUU motif of the Ur element RNA oligo.