Effect of hypoxia on the structure of HIF1A mRNA 5ʹ untranslated region
Faculty Mentor(s) Name(s)
Arnab Sengupta
Abstract
The HIF1A gene is a transcriptional activator that regulates cellular responses to hypoxia by inducing transcription in many other genes, and plays an essential role in embryonic vascularization, tumor angiogenesis, and ischemic diseases. Overexpressed HIF1A gene has been found to be linked to many forms of carcinoma, breast, ovarian, and lung cancer. The gene is reported to exhibit internal ribosome entry site (IRES) activity. IRES activity uses regulatory structures located in the 5ʹ untranslated region of the mRNA allowing cap-independent translation. In the case of the HIF1A mRNA, IRES activity must be specifically activated under hypoxic stress. The mechanism of this process is unclear. To address this, we are interested in measuring changes in the structure and interactions of the regulatory region subjected to chemically-induced hypoxia. We have applied SHAPE-MaP to investigate the secondary structure of the regulatory region of the HIF1A mRNA in human cell lines to better understand the mechanisms of stress-induced initiation. We compare SHAPE data collected using gene-specific targeting of the HIF1A mRNA under (a) cell-free, (b) in-cell normoxic, and (c) in-cell hypoxic conditions. Our cell-free structure model reveals a highly structured 5ʹ UTR with multiple short hairpins motifs. Next, we closely compare live cell SHAPE data identifying regions that are affected by hypoxia. In addition to changes in SHAPE reactivity, we have detected changing patterns of protein interactions within the 5ʹ UTR under the varying conditions. Future directions include utilizing RNP-MaP, a UV-crosslinking strategy to examine specific protein-motif interactions affecting hypoxic translation, along with DMS-MaP to directly evaluate base-pairing with more specificity. Lastly, we identify orthogonal strategies to validate the HIF1A mRNA structure and interactions.
Start Date
27-3-2024 10:20 AM
End Date
27-3-2024 10:28 AM
Location
Arts and Sciences 2-70
Effect of hypoxia on the structure of HIF1A mRNA 5ʹ untranslated region
Arts and Sciences 2-70
The HIF1A gene is a transcriptional activator that regulates cellular responses to hypoxia by inducing transcription in many other genes, and plays an essential role in embryonic vascularization, tumor angiogenesis, and ischemic diseases. Overexpressed HIF1A gene has been found to be linked to many forms of carcinoma, breast, ovarian, and lung cancer. The gene is reported to exhibit internal ribosome entry site (IRES) activity. IRES activity uses regulatory structures located in the 5ʹ untranslated region of the mRNA allowing cap-independent translation. In the case of the HIF1A mRNA, IRES activity must be specifically activated under hypoxic stress. The mechanism of this process is unclear. To address this, we are interested in measuring changes in the structure and interactions of the regulatory region subjected to chemically-induced hypoxia. We have applied SHAPE-MaP to investigate the secondary structure of the regulatory region of the HIF1A mRNA in human cell lines to better understand the mechanisms of stress-induced initiation. We compare SHAPE data collected using gene-specific targeting of the HIF1A mRNA under (a) cell-free, (b) in-cell normoxic, and (c) in-cell hypoxic conditions. Our cell-free structure model reveals a highly structured 5ʹ UTR with multiple short hairpins motifs. Next, we closely compare live cell SHAPE data identifying regions that are affected by hypoxia. In addition to changes in SHAPE reactivity, we have detected changing patterns of protein interactions within the 5ʹ UTR under the varying conditions. Future directions include utilizing RNP-MaP, a UV-crosslinking strategy to examine specific protein-motif interactions affecting hypoxic translation, along with DMS-MaP to directly evaluate base-pairing with more specificity. Lastly, we identify orthogonal strategies to validate the HIF1A mRNA structure and interactions.