The Impact of Two RNA Binding Proteins, FMRP and FUS, on a Protein Adapter Complex in Neurons Restricted; Files Only
Sun, Jiarong (Spring 2024)
Abstract
Protein homeostasis, vital for cellular function, is intricately regulated in mammals through transcriptional, translational, and degradation mechanisms. RNA binding proteins (RBPs) are central to this regulation, governing protein synthesis, mRNA transport, and RNA processing in neurons. Dysregulation of RBPs, such as Fragile X Messenger Ribonucleoprotein (FMRP) in Fragile X syndrome (FXS) and Fused in Sarcoma (FUS) in Amyotrophic lateral sclerosis-Frontotemporal dementia (ALS-FTD), disrupts proteostasis. Depletion of FMRP alters surface expression of key neuronal membrane proteins bidirectionally, hinting at regulatory mechanisms beyond translation repression. We hypothesize that FMRP maintains proper surface protein level of several membrane proteins through regulating clathrin mediated endocytosis (CME), since studies from our lab and others showed that the mRNA encoding clathrin adapter complex AP2 subunits are targets of FMRP mediated translational repression. In this thesis, we demonstrated reduced total surface protein level in Fmr1 knockdown cortical neuron cultures analyzed by Western blotting and immunofluorescent staining. Plasma membrane enriched samples were prepared for a proteomics study and identified many altered protein candidates including elevated AP2 subunits. As FUS is also known to bind and regulated mRNAs encoding AP2 subunit, we sought to investigate potential co-regulation of AP2 by FMRP and FUS. RNA immunoprecipitation (RIP) experiments revealed that FUS could bind to AP2 subunit mRNA, which competed with FMRP association with AP2A1 mRNA. The AP2 protein level was examined by Western blotting and individual cell image analysis, which revealed a reverse trend in AP2 protein levels when FUS is overexpressed with and without endogenous FMRP. These experiments show that FMRP is necessary for the ability of FUS to reduce the expression of AP2. Based on our findings, we suggest that FUS cooperates with FMRP to suppress AP2 synthesis, while in the absence of FMRP, FUS enhances AP2 expression possibly by potentially stabilizing the mRNA. More work is needed to investigate the molecular mechanisms for FMRP and FUS to regulate AP2 mRNA. This thesis underscores the pivotal role of RBPs in neuronal protein expression, utilizing FXS and ALS as disease models. Future research targeting protein homeostasis restoration holds promise for addressing autism and related disorders.
Table of Contents
Table of Contents
I. Introduction .........................................................................8
II. Methods ...............................................................................17
III. Results ................................................................................21
IV. Discussion ..........................................................................26
V. Figures .................................................................................30
VI. References .........................................................................38
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File download under embargo until 24 May 2026 | 2024-04-12 21:48:19 -0400 | File download under embargo until 24 May 2026 |
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