Molecular mechanisms for the post-transcriptional regulation of tissue factor expression
File(s)
Author(s)
Chatterton-Bartley, Courtney
Type
Thesis
Abstract
Tissue Factor (TF) plays vital roles in many cellular processes involved in angiogenesis, wound healing, intracellular signalling, tumour metastasis, inflammation, haemostasis, and thrombosis. It is known to be regulated at the transcriptional and post-transcriptionally levels, the latter being controlled in large part by RNA binding proteins (RBPs). Understanding the post-transcriptional regulation of TF could help with the design of treatment to control thrombosis without impairing haemostasis.
RBPs can control post-transcriptional regulation by binding to the 3’-UTR of their target mRNA. About 34 million years ago during evolution an inverted Alu element inserted into the TF 3’-UTR. The insertion of the Alu element potentially introduced more binding sites for RBPs, and we hypothesised this altered post-transcriptional control.
Using RNA pull-down assays and mass spectrometry, this project discovered that the Alu element insertion considerably changes proteins that interact with the TF 3’-UTR. For example, Human Antigen R (HuR) binding is much increased, and attributable experimentally to a poly-U tract at the 5’ junction of the Alu insertion. HuR has many roles including in mRNA stability, mRNA degradation, and protein trafficking.
Using fluoroprobe constructs regulated by the TF 3’-UTR with or without the Alu insertion, we found with confocal microscopy that the Alu element enhances protein localisation to the Golgi (p < 0.0482), and the absence of the Alu element led to more localisation to the cell membrane (p < 0.0159). We propose that the insertion of the Alu element into the TF 3’-UTR during evolution allowed more TF to be stored in the Golgi, ready for rapid mobilisation when required.
RBPs can control post-transcriptional regulation by binding to the 3’-UTR of their target mRNA. About 34 million years ago during evolution an inverted Alu element inserted into the TF 3’-UTR. The insertion of the Alu element potentially introduced more binding sites for RBPs, and we hypothesised this altered post-transcriptional control.
Using RNA pull-down assays and mass spectrometry, this project discovered that the Alu element insertion considerably changes proteins that interact with the TF 3’-UTR. For example, Human Antigen R (HuR) binding is much increased, and attributable experimentally to a poly-U tract at the 5’ junction of the Alu insertion. HuR has many roles including in mRNA stability, mRNA degradation, and protein trafficking.
Using fluoroprobe constructs regulated by the TF 3’-UTR with or without the Alu insertion, we found with confocal microscopy that the Alu element enhances protein localisation to the Golgi (p < 0.0482), and the absence of the Alu element led to more localisation to the cell membrane (p < 0.0159). We propose that the insertion of the Alu element into the TF 3’-UTR during evolution allowed more TF to be stored in the Golgi, ready for rapid mobilisation when required.
Version
Open Access
Date Issued
2023-11-03
Date Awarded
01/01/2024
License URL
Advisor
Haskard, Dorian
Boyle, Joe
Sponsor
The Berenblut Scholarship
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)