John Pum

RNA binding proteins regulate gene expression by binding and affecting the stability, localization, and translation of their mRNA targets. The human Pumilio RNA binding proteins, PUM1 and PUM2, regulate thousands of genes with many linked to cancer. PUMs predominantly downregulate genes; however, some genes are activated by PUMs. I hypothesize that PUM1 and PUM2 activate specific targets through interactions with putative cofactors that associate with activation element(s) and influence PUM regulation. To test this, I created luciferase-based assays utilizing a recombinant nanoluciferase plasmid (pNLP) fused to the 3’UTR of the gene CREBL2. CREBL2 contains two Pumilio response elements (PRE) in the 3’UTR, one of which is repressed and the other activated by PUMs. First, the luciferase-based assays were optimized using various concentrations of pNLP and firefly (ff) control plasmid transfections into HEK293 cells. Then, to identify a potential activation element, minimum constructs of the 3’UTR of CREBL2 consisting of 50 base pairs flanking each side of each PRE sequence were introduced into the pNLP reporter. The minimum constructs yielded results of 55% repression relative to the control for the first PRE minimum and 20% activation relative to the control for the second PRE minimum sequence. This suggests that an activation element may reside within the second minimum sequence. I am currently using Gibson assembly to create a reporter to test the full length CREBL2 gene, now including the 5’UTR and coding region, to identify possible activation sites outside of the 3’UTR.