Vergara, Kevin
(2025)
Exploring the role of LUCAT1 and RBMX in human macrophages.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Long noncoding RNAs (lncRNA) and RNA binding proteins (RBP) impact nearly all biological responses. lncRNAs are transcripts larger than 200 nucleotides that play crucial roles in many cells and tissues by regulating gene expression. RBPs act in parallel and in concert with lncRNA to mediate the fate of RNA. Here we sought to determine the mechanism of action of LUCAT1, a lncRNA, discovered by us to be an important regulator of the macrophage inflammatory response. LUCAT1 promotes the expression of many immune- and inflammation-associated genes including CXCL7, IL-24 and NT5E at the transcriptional level. Using in vitro binding assays with biotinylated LUCAT1 RNA and nuclear lysates, we have identified HNRNPL and MATRIN3 as potential LUCAT1-ineracting proteins. siRNA knockdown of putative LUCAT1-interacting proteins revealed that RBMX promotes expression of LUCAT1 and LUCAT1-regulated genes. RBMX is a nuclear protein expressed in all cells and plays an important role by binding to RNA and regulating splicing, transcription, and stability. Dysregulation of RBMX expression is linked to cancer and neurological disorders, but its function in immune cells is unknown. To uncover the cellular functions of RBMX in human monocytes and macrophages, we used CRISPR/Cas9 editing to generate a monoclonal THP-1 cell line with an in frame internal deletion in RBMX spanning amino acid positions 218-250 (RBMXΔ218-250). Immunofluorescence in PMA-differentiated THP-1 macrophages shows that RBMXΔ218-250 is localized in the nucleus but punctate structures are smaller and diffused compared to WT RBMX. Upon PMA stimulation, RBMXΔ218-250 cells exhibit a defect in macrophage-like morphology. Further, LPS stimulation of PMA-differentiated RBMXΔ218-250 cells resulted in attenuated ERK1/2 signaling. Transcriptome analysis revealed decreases in growth factors and other cytokines in resting and stimulated RBMXΔ218-250 cells compared to control. These results provide insights into the function, mechanism, and potential cooperative relationship of LUCAT1 and RBMX in macrophages.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
18 February 2025 |
| Date Type: |
Publication |
| Defense Date: |
10 September 2024 |
| Approval Date: |
18 February 2025 |
| Submission Date: |
18 December 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
125 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Microbiology and Immunology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
LUCAT1, lncRNA, RNA, RBMX, macrophage, monocyte |
| Date Deposited: |
18 Feb 2025 17:43 |
| Last Modified: |
18 Feb 2025 17:43 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/47307 |
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