Immunology/Cancer Biology

Malone Research Laboratory: Ruchika Loya
Teitell Research Laboratory at UCLA: Rhine Shen

“ Regulation of the PNPase gene promoter.”

How genes are regulated in different cell types, such as skin or bone, and turned on/off in normal development is poorly understood. The goal of this project is to isolate and characterize the minimal or core promoter region that controls expression of a mitochondrial protein involved in anti-cancerous effects, called Mammalian Phospho-ribonuclease (PNPase), using molecular biology techniques in cells ideally suited for answering this fundamental question.

Precisely controlling when and where genes are turned on and off, known as controlling gene expression, is necessary for all cells to develop and function normally and avoid becoming cancerous or dying prematurely. PNPase is localized in the mitochondria has ribonuclease and poly (A) polymerase activities. Interestingly, PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates pro-inflammatory cytokine production; all of these are characteristic of anti cancerous effects or tumor suppression. The human and mouse PNPase promoter sequence has been identified between -1500bp from the start of transcription including the 200bp 5’ untranslated region.  Bioinformatics have identified several putative transcription factor binding sites.  The PNPase promoter appears to be a TATA-less and INR-less promoter.  Definitive confirmation of this possibility remains to be resolved.

A preliminary deletion series of both the human and mouse PNPase promoters have been generated by PCR and subcloned into the pGL3–luciferase vector: 1kb, 750 bp, 400bp, and 250 bp promoter fragments. Further deletional analyses will identify the minimal promoter fragment and site-directed mutagenesis of the identified minimal PNPase promoter construct will then be used to determine the functional regulatory elements involved in PNPase gene regulation.

Appropriately controlling when and where the PNPase gene is expressed is critical for the anti-cancer effects it provides to the cell.