Nuclear binding and translation regulation by FMRP through novel associated protein and putative RNA helicase MOV10

The work presented in this dissertation focuses primarily on the role of FMRP, the fragile X mental retardation protein and its role in translational regulation. More specifically, I began my studies trying to understand the role of FMRP in the nucleus. This work is novel and interesting because the nuclear role of FMRP has not been widely studied. FMRP is an RNA binding protein that is important for normal neuronal development and maturation as well as proper cognitive development as the lack of FMRP results in fragile X syndrome (FXS). I next move out of the nucleus and study the role of FMRP and translation in the cytoplasm. This work elaborates on a novel FMRP associated protein, putative RNA helicase MOV10 Chapter 1 begins with an introduction to fragile X syndrome as well as a current literature review of the fragile X field. I introduce the history of fragile X syndrome and the characteristics of the syndrome as well as introducing the key player: FMRP. FMRP is an interesting protein because it has several RNA binding domains as well as several post translational modifications. Despite all that is currently known about FMRP, there is still a large gap in understanding about how these modifications act as guides for specific RNA targeting and binding. My first project outlined in Chapter 2 focuses on the role of FMRP in the nucleus. FMRP contains both a Nuclear Localization Sequence (NLS) as well as a Nuclear Export Sequence (NES), however how FMRP enters the nucleus is unclear because of its non-canonical NLS. I demonstrate that FMRP enters the nucleus and that it requires Tap/Nxf1 for efficient export from the nucleus. Additionally, I use the Xenopus laevis oocyte system to demonstrate that FMRP is indeed present along nascent mRNA transcripts suggesting that FMRP enters the nucleus and can target newly transcribed RNAs. Next, in Chapter 3, I focus on a novel FMRP associated protein, MOV10. MOV10 is a putative RNA helicase that associates with FMRP. I demonstrate that MOV10 and FMRP associate in a salt and RNA dependent manner and that the two proteins exist in tissue in similarly sized granules suggesting a cellular interaction. We also determined that FMRP requires MOV10 to recruit a number of brain specific mRNAs and that both FMRP and MOV10 bind G quadruplex structures specifically. Using iClip, we identified MOV10 specific mRNA targets as well as identifying binding sites of MOV10. We also found that Ago2 binding sites influenced the fate of MOV10 clip targets, suggesting a role of MOV10 acting as a facilitator or inhibitor of miRNA mediated regulation depending on proximity to Ago2 binding sites. Chapter 4 develops a novel single molecule study directed towards understanding how MOV10 behaves as a helicase. I generated mRNA constructs to study G quadruplex forming RNA sequences at the single molecule level and establish that sc1and sc1 mutant RNAs behave different in salt solutions. In addition to studying sc1 and sc1 model RNAs, I expanded the study to encompass hASH1, a recently identified FMRP mRNA target that contained tandem GQ sequences near the 5’ cap, and also PSD-95, a well established FMRP target. Through the efforts of this chapter, it will be possible to continue to further elucidate the behavior of MOV10 on target mRNAs identified through the iCLIP studies. In Chapter 5, I explore the role of MOV10 in the 5’UTR of mRNA targets. Chapter 3 focused more on the role of the 3’UTR and miRNA mediated translational regulation and mRNA stability. From the data generated from the iCLIP studies, we found a number of genes that are bound by MOV10 in the 5’UTR. Using hASH1 as a model mRNA target based on previously published work on FMRP and hASH1 translation, we found that upregulation of hASH1 by FMRP is dependent on the presence of MOV10. These studies demonstate that MOV10 may also play a significant role in translation through the 5’UTR through a mechanism that is independent of the miRNA pathway