Stress-induced changes in 5ʹ end regulatory structures of the TP53 mRNA
Faculty Mentor(s) Name(s)
Dr. Arnab Sengupta
Abstract
Tumor suppressor TP53 is responsible for multiple regulatory functions including DNA repair, apoptosis, and cell cycle control. Loss-of-function mutations of the TP53 gene have been found in about 50% of all human cancers. Expression of the TP53 gene is regulated using highly diverse mechanisms with 13 known isoforms. Interestingly, the TP53 mRNA reportedly uses internal ribosome entry site (IRES)-mediated translation under cellular stress. Previous studies have implicated the role of structurally stable motifs in the 5ʹ untranslated region for mediating cap- independent translation. We present a secondary structure model of the TP53 5ʹ regulatory region. This region includes an alternate downstream start codon. Our model, based on SHAPE- MaP data from gently extracted total RNA of the A549 human lung carcinoma cell line using 5- nitroisatoic anhydride (5NIA), confirms previously observed secondary structure motifs. We then apply 5NIA to probe RNA in-cell to detect changes in SHAPE reactivity in live cells and compare reactivities with the cell-free model. We observe that the IRES-related motifs remain stable in unstressed live cells. Next, we treat cells with etoposide, an oncogenic stress inducer. Our initial studies detect measurable changes in SHAPE reactivity in a short stem-loop motif downstream of the standard AUG start codon. Prior reports indicate the role of IRES-transacting factors (ITAFs) including Hdm2 interacting with this mRNA region, and in certain cases correlating with the translation of an N-terminal truncated TP53. We outline future directions investigating the TP53 mRNA structure under different stress conditions, and also aim to identify changes in ITAF-interaction sites using RNP-MaP.
Start Date
27-3-2024 3:30 PM
End Date
27-3-2024 3:38 PM
Location
Arts and Sciences 2-70
Stress-induced changes in 5ʹ end regulatory structures of the TP53 mRNA
Arts and Sciences 2-70
Tumor suppressor TP53 is responsible for multiple regulatory functions including DNA repair, apoptosis, and cell cycle control. Loss-of-function mutations of the TP53 gene have been found in about 50% of all human cancers. Expression of the TP53 gene is regulated using highly diverse mechanisms with 13 known isoforms. Interestingly, the TP53 mRNA reportedly uses internal ribosome entry site (IRES)-mediated translation under cellular stress. Previous studies have implicated the role of structurally stable motifs in the 5ʹ untranslated region for mediating cap- independent translation. We present a secondary structure model of the TP53 5ʹ regulatory region. This region includes an alternate downstream start codon. Our model, based on SHAPE- MaP data from gently extracted total RNA of the A549 human lung carcinoma cell line using 5- nitroisatoic anhydride (5NIA), confirms previously observed secondary structure motifs. We then apply 5NIA to probe RNA in-cell to detect changes in SHAPE reactivity in live cells and compare reactivities with the cell-free model. We observe that the IRES-related motifs remain stable in unstressed live cells. Next, we treat cells with etoposide, an oncogenic stress inducer. Our initial studies detect measurable changes in SHAPE reactivity in a short stem-loop motif downstream of the standard AUG start codon. Prior reports indicate the role of IRES-transacting factors (ITAFs) including Hdm2 interacting with this mRNA region, and in certain cases correlating with the translation of an N-terminal truncated TP53. We outline future directions investigating the TP53 mRNA structure under different stress conditions, and also aim to identify changes in ITAF-interaction sites using RNP-MaP.