•  
  •  
 

STRESS-INDUCED CHANGES IN THE 5' END REGULATORY STRUCTURES OF THE TP53 mRNA

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 cases of human cancer. Expression of TP53 is regulated using highly diverse mechanisms resulting in 13 known isoforms. Interestingly, TP53 mRNA has been reported to use IRES-mediated translation, especially under cellular stress. Previous studies have implicated the role of structurally stable motifs in the 5’UTR region of TP53 among other genes for mediating cap-independent translation. We present a secondary structure model of the regulatory region spanning 142 nucleotides upstream and 356 nucleotides downstream of the start codon for full-length TP53. The target region includes an alternate downstream start codon. Our model, based on selective 2¢ hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) data from gently extracted total RNA from A549 human lung carcinoma cell line using 5NIA, confirms previously observed secondary structure motifs such as stem-loops from differing techniques. We then apply the in-cell probing reagent 5NIA to detect changes in SHAPE reactivity in live A549 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 hairpin motif downstream of the standard AUG start codon. Furthermore, base-pairing probabilities of this motif region drop from >80% to <30% under etoposide stress. Prior reports implicate 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.

Acknowledgements

NSF

This document is currently not available here.

Share

COinS