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Identifying RNA-binding landscapes of PXR using enhanced UV-crosslinking and immunoprecipitation followed by sequencing

Wang, Xiaofei (2021) Identifying RNA-binding landscapes of PXR using enhanced UV-crosslinking and immunoprecipitation followed by sequencing. Master's Thesis, University of Pittsburgh. (Unpublished)

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Abstract

Pregnane X Receptor (PXR) is a nuclear receptor whose primary function is to sense the presence of foreign toxic substances and subsequently up-regulate the expression of proteins involved in the clearance of these substances transcriptionally. Intriguingly, emerging evidence suggests that PXR may also bind with RNAs and directly regulate them. For example, PXR was reported to decrease the stability of TLR4 mRNA after treated with its ligand. However, the underlying mechanism of the PXR’s regulation of RNAs remains elusive. Here, we aim to provide insights into the post-transcriptionally regulatory function of PXR. Specifically, we used enhanced UV-crosslinking and immunoprecipitation (eCLIP) to identify the RNAs that can bind with PXR in both colorectal cancer cell lines and mouse livers with humanized PXR. This analysis revealed that PXR can bind with thousands of mRNAs in both human colorectal cancer cell lines and mouse livers. Moreover, analysis of PXR’s binding location demonstrated that PXR eCLIP peaks are highly enriched in the 3'UTR region of mRNAs. Treatment of rifampicin, a ligand of PXR, can further increase PXR binding preference in mRNA 3’UTR. Genes with PXR binding to their 3’UTR were enriched in several pathways like cell migration and apoptosis, metabolism of glucose and triglyceride, and vascular functions, which are all known to be related to PXR functions. Since 3’UTR is responsible for RNA stability, it is possible that PXR can post-transcriptionally regulate these signaling pathways. In this regard, we detected the correlation between the expression of PXR and its eCLIP targets using the TCGA database. PXR expression level are positively corrected with some of its eCLIP-seq targets like VEGFA and Igf1, suggesting PXR may regulate VEGFA and Igf1 expression post-transcriptionally. In summary, our study suggests PXR can bind to the 3'UTR of mRNAs and regulate their expression level post-transcriptionally.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Wang, XiaofeiXIW141@pitt.eduXIW1410000-0002-9415-5294
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairYang, Dadyang@pitt.edu
Committee MemberZhang, Minmiz45@pitt.edu
Committee MemberXie, Wenwex6@pitt.edu
Committee MemberMa, Xiaochaomxiaocha@pitt.edu
Date: 19 April 2021
Date Type: Publication
Defense Date: 2 April 2021
Approval Date: 19 April 2021
Submission Date: 6 April 2021
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 37
Institution: University of Pittsburgh
Schools and Programs: School of Pharmacy > Pharmaceutical Sciences
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: PXR, eCLIP-seq, RNA stability
Date Deposited: 19 Apr 2021 15:12
Last Modified: 19 Apr 2021 15:12
URI: http://d-scholarship.pitt.edu/id/eprint/40514

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