Fathia Elmoghe
LSAMP Scholar
Major: Biology
Mentors: Heather Hundley, Priyanka Mukherjee, and Ananya Mahapatra
Glioblastoma is the deadliest form of brain cancer, with a median survival rate of 20 months and near inevitable recurrence. All cancers including glioblastoma are the result of an uncontrolled proliferation of cells which is caused due to altered gene expressions in different genes. ADAR proteins (Adenosine Deaminases Acting on RNAs) are RNA binding proteins which regulate gene expression through either binding to or editing of RNA (turning Adenosine to Inosine, also known as A-to-I editing), causing a change in the sequence of messenger RNA (mRNA) and ultimately affecting protein structure and consequently, function. Previous studies have concluded that ADAR proteins are essential to proper neural function and have also shown that the expression of ADAR3 specifically (which is only found in brain tissue) is higher in glioblastoma than in normal brain tissues. Previously in the Hundley lab,1435 ADAR3 bound targets were identified from U373 having endogenous expression of ADAR3. Interestingly of those bound transcripts, 30% are long noncoding RNA, which includes Antisense RNAs. This prompted us to question whether ADAR3 regulates gene expression profiles of cancer-related genes by regulating Antisense Transcripts(AS)? Preceding studies have also shown that ADAR3 negatively regulates GRIA2 editing mediated by ADAR2 by competing for binding to Q/R site of GRIA2 in the U87 cell line. This brought us to the inquiry of how does the altered expression of ADAR3 affect the binding and editing ability of ADAR1? Quantitative PCR was conducted to see if either the Antisense transcript or the complementary Sense gene transcript is differentially expressed. RNA was isolated from a U373 cell line that expressed control and ADAR3-expressing plasmid. We found that the INKA2 Sense gene and the INKA2 Antisense gene were not differentially expressed in the presence of ADAR3. However, we did find that the BCAN Antisense gene was differentially expressed in the presence of ADAR3. Epitope tagged ADAR1-V5 was cloned into mammalian expression plasmid through a Polymerase Chain Reaction (PCR).The product was then transformed in chemically
competent E.coli cells, then independent colonies were isolated and grown in LB media containing Ampicillin selection. Retrovirus was then extracted and transduced into U87
cell line, and then selected for retroviral infected cells with G418 puromycin. The expression of V5-ADAR1 in U87 control cells and U87 cells expressing 3X FLAG tagged ADAR3 was confirmed by immunoblotting. A future implication of these findings is that a cell line could be generated wherein ADAR1 and ADAR3 can regulate gene expression together.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. IN LSAMP is supported by NSF Award Number: HRD-1618408, 2016-2022.
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