Fragile X Syndrome (FXS) is a neurodevelopmental disorder that is the most prevalent form of inherited mental retardation and the primary monogenetic cause of autism. FXS, as well as some manifestations of autism spectrum disorder, results from improper RNA regulation due to a deficiency of fragile X mental retardation protein (FMRP) in neurons. FMRP and its autosomal paralogs, fragile X related proteins 1 & 2 (FXR1P/2P), have been implicated in many aspects of RNA regulation, from protein synthesis to mRNA stability and decay. The literature on the fragile X related proteins’ (FXPs) role in mRNA regulation and their potential mRNA targets is vast, yet there is little overlap between mRNA targets identified, or the proposed mechanism of mRNA regulation. There is great interest in studying this family of proteins, yet researchers have faced much difficulty in expressing and purifying the full-length versions of these proteins in sufficient quantities. We developed a simple, rapid, and inexpensive procedure that allows for the recombinant expression and purification of full-length human FMRP, FXR1P, and FXR2P from Escherichia coli in high yields, free of protein and nucleic acid contamination. After confirming each protein’s identity with mass spectrometry, we assessed the proteins’ function after purification, and confirmed their binding to pseudoknot and G-quadruplex forming RNAs as well as their ability to regulate translation in vitro. After developing a method to successfully purify the FXPs, we investigated how each protein regulated the translation of various mRNAs in a simplified in vitro translation (IVT) system. We developed an approach to investigate the function of FXPs in translational control using three potential mRNA targets. Briefly, we first selected top mRNA candidates found to be associated with the FXPs and whose translation are influenced by one or more of the FXPs. We then narrowed down the FXPs’ binding site(s) within the mRNA and analyzed the strength of this binding in vitro. Finally, we determined how each FXP affects the translation of a minimal reporter mRNA containing the FXP-binding site in a minimalistic IVT system we designed. Overall, we observed all FXPs to bind RNAs containing G-quadruplexes with high affinity, for example, cyclin dependent kinase inhibitor p21 and FMRP’s own coding region. Interestingly, FMRP inhibited the translation of each mRNA differently, in a manner that appears to correlate with its binding affinity for each mRNA. In contrast, FXR1P/2P inhibited all mRNAs tested. Finally, although binding of these RNAs was due to the RGG (arginine-glycine-glycine) motif-containing C-terminal region of the FXPs, this region was not sufficient to cause inhibition of translation. While designing a minimal IVT system, we discovered a novel RNA target of the FXPs. Although the most well-known RNA structural target of this family of proteins is the G-quadruplex (GQ), we discovered a non-GQ forming RNA target that is about 100 nucleotides in length and binds to all three FXPs with nanomolar dissociation constants. Furthermore, we determined that the last 102 amino acids of FMRP, which includes the RGG motif, were necessary and sufficient to bind this novel RNA target. To the best of our knowledge, this is the first example of a non-GQ structure RNA to be bound by the RGG motif/C-termini of FMRP.