Investigating miRNA Regulation of the Human APOBEC3 Enzymes
Start Date
15-4-2021 2:00 PM
End Date
15-4-2021 3:00 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Allison Land
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Human cells have evolved numerous ways to defend themselves against viral invaders, one example is the human APOBEC3 (A3) class of enzymes. This family of proteins consist of seven enzymes, A3A, B, C, D, F, G and H; which function as cytosine deaminases. By mutating viral DNA, the enzymes can hinder or stop replication of viruses such as human papillomavirus, herpes simplex virus, and HIV 1. Several of the A3 enzymes have also been implicated in contributing to cancers such as head and neck and breast cancers by mutating cellular genomic DNA, making the ability to control A3 expression an attractive target for cancer therapy. The A3 enzymes are regulated by small fragments of RNA called miRNA. I am currently working on developing an assay to test A3 enzymes and the regulatory effects of their miRNAs in the human cell line HeLa. I hypothesize that the A3 family is regulated by miRNAs; each member by a different repertoire of miRNAs. To test this hypothesis A3 3’ UTRs were inserted into psiCHECK-2 vectors that encode both firefly and sea pansy luciferase. Potential miRNAs were compared and sorted by their predicted efficiency of interacting with the A3 3’ UTRs. Candidate miRNAs will be cloned into pcDNA 3.1 expression vectors. The miRNA and A3 3’ UTR plasmids will be co-transfected into HeLa cells and the expression of the two luciferase proteins will be measured to quantify the miRNA regulation of the A3 3’ UTRs. After testing the A3 3’ UTR constructs and miRNAs I will mutate base pairs on the miRNA to confirm that the regulatory effect is from the miRNAs.
Investigating miRNA Regulation of the Human APOBEC3 Enzymes
Human cells have evolved numerous ways to defend themselves against viral invaders, one example is the human APOBEC3 (A3) class of enzymes. This family of proteins consist of seven enzymes, A3A, B, C, D, F, G and H; which function as cytosine deaminases. By mutating viral DNA, the enzymes can hinder or stop replication of viruses such as human papillomavirus, herpes simplex virus, and HIV 1. Several of the A3 enzymes have also been implicated in contributing to cancers such as head and neck and breast cancers by mutating cellular genomic DNA, making the ability to control A3 expression an attractive target for cancer therapy. The A3 enzymes are regulated by small fragments of RNA called miRNA. I am currently working on developing an assay to test A3 enzymes and the regulatory effects of their miRNAs in the human cell line HeLa. I hypothesize that the A3 family is regulated by miRNAs; each member by a different repertoire of miRNAs. To test this hypothesis A3 3’ UTRs were inserted into psiCHECK-2 vectors that encode both firefly and sea pansy luciferase. Potential miRNAs were compared and sorted by their predicted efficiency of interacting with the A3 3’ UTRs. Candidate miRNAs will be cloned into pcDNA 3.1 expression vectors. The miRNA and A3 3’ UTR plasmids will be co-transfected into HeLa cells and the expression of the two luciferase proteins will be measured to quantify the miRNA regulation of the A3 3’ UTRs. After testing the A3 3’ UTR constructs and miRNAs I will mutate base pairs on the miRNA to confirm that the regulatory effect is from the miRNAs.
