Mihaela Rita MihailescuInterim Chair and Professor
Bayer School of Natural and Environmental Sciences
Chemistry & Biochemistry
308B Mellon Hall
Education:B.S., M.S. University of Bucharest
Ph.D., Wesleyan University, CT
Post Doctoral Fellow, Center for Advanced Research in Biotechnology
Fragile X syndrome (FXS), the most common form of inherited mental impairment, affects 1 in 3,600 to 4,000 males and 1 in 4,000 to 6,000 females. In the vast majority of cases, FXS is caused by an unstable expansion of a CGG trinucleotide repeat in the 5' untranslated region (UTR) of the fragile X mental retardation-1 (Fmr1) gene. The general population has 6-54 CGG repeats, which expand to 55-200 in the Fmr1 premutation carriers, and exceed 200 in the full mutation, which causes the Fmr1 gene silencing and loss of expression of the fragile X mental retardation protein (FMRP).
FMRP has two types of RNA-binding motifs (two K Homology domains and an arginine-glycine-glycine (RGG) rich box), a nuclear localization signal (NLS) at its N-terminus and a nuclear export signal (NES) at its C-terminus. FMRP is subject to the posttranslational modifications of phosphorylation in a region N terminal to its RGG box and arginine methylation within the RGG box. Additionally, the Fmr1 gene, which has 17 exons, can undergo alternative splicing involving the exons 12 and 14 and the choice of acceptor sites in exons 15 and 17.
FMRP is involved in the transport and translation regulation of specific neuronal mRNA targets, and although it is assumed that posttranslational modifications and alternative splicing events might mediate these functions, the detailed mechanisms by which the protein accomplishes these functions remain elusive. There is no clear consensus in the literature on the FMRP RNA recognition motifs, but the G quadruplex (GQ) structure has been proposed early on as a specific motif bound with high affinity and specificity by the FMRP RGG box. Our laboratory has validated the existence of GQ structures in many FMRP neuronal mRNA targets and characterized the thermodynamics of their interactions with FMRP.
With respect to its translation regulator function, FMRP has been shown to repress the translation of a subset of its mRNA targets by working in conjunction with the microRNA (miRNA)-guided RNA induced silencing complex (RISC). miRNAs are small non-coding RNAs ~22 nucleotides (nt) long produced through the cleavage of larger 60-110 nt precursor miRNAs (pre-miRNAs) by the enzyme Dicer and associated proteins. Upon its incorporation into RISC, miRNA guides it to its target mRNA, where depending on the miRNA level of complementarity to the mRNA, RISC represses translation or induces the mRNA degradation. In a few cases, FMRP has been shown to directly affect the translation of specific mRNAs through its interactions with RISC. FMRP facilitates the interactions of the miR125a-loaded RISC with the post-synaptic density 95 (PSD-95) mRNA to regulate its translation. Specifically, when FMRP is phosphorylated RISC is associated with PSD-95 mRNA, repressing its translation, whereas upon FMRP de-phosphorylation in response to synaptic input, RISC dissociates allowing for the PSD-95 protein synthesis. The mechanisms by which this switch is mediated are not known. miR-125a has its binding site embedded in a G rich region, which we showed forms GQ structures that modulate the accessibility of the RISC complex. There are other FMRP associated miRNAs whose demonstrated target mRNAs have the potential to form GQ structures either within the miRNA binding site or in its close proximity, but their role in the RISC-mediated translation regulation is not known. Additionally, there are other FMRP target mRNAs that have miRNA binding sites within G rich regions that we showed form GQ structures recognized by FMRP.
Thus, our laboratory uses biochemical and biophysical methods to try to elucidate the molecular mechanisms by which FMRP interacts with the miRNA pathway to exert its translation regulator function and how alternative splicing and FMRP posttranslational modifications might affect the protein function.
Imperatore J.A., Then M.L., McDougal K.B. & Mihailescu M.R., Characterization of a G-quadruplex structure in pre-miRNA-1229 and in its Alzheimer's disease-associated variant rs2291418: implications for miRNA-1229 maturation. Int J Mol Sci. 2020 Jan 24;21(3):767.
Imperatore J.A., McAninch D.S., Valdez-Sinon, AN, Bassell, G.J. & Mihailescu, M.R., FUS Recognizes G Quadruplex Structures Within Neuronal mRNAs. Front Mol Biosci 2020 Feb 7;7:6. doi: 10.3389/fmolb.2020.00006. eCollection 2020. (invited article)
DeMarco B., Stefanovic S., Williams A., Moss, K.R., Anderson B.R., Bassell G.J. & Mihailescu M.R., FMRP-G quadruplex mRNA- miR-125a interactions: Implications for miR-125a-mediated translation regulation of PSD-95 mRNA. PLoS One 2019 May 21;14(5): e0217275. doi: 10.1371/journal.pone.0217275. eCollection 2019.
McAninch, D.S., Heinaman A.M., Lang, C.N., Moss, K.R., Bassell, G.J., Mihailescu, M.R. & Evans, T.L., Fragile X mental retardation protein recognizes a G quadruplex structure within the survival motor neuron domain containing 1 mRNA 5'-UTR. Mol. Biosyst. 2017, 13(8):1448-1457.
Bartley, C.M., O'Keefe, R.A., Blice-Baum, A., Mihailescu, M.R., Miyares, L., Karaca, E. & Bordey, A., Mammalian FMRP S499 is phosphorylated by CK2 and promotes secondary phosphorylation of FMRP, eNeuro 2016 3(6). pii: ENEURO.0092-16.2016. eCollection 2016 Nov-Dec.
Williams, K.R., McAninch, D.S., Stefanovic, S., Xing, L., Allen, M., Li, W., Feng, Y., Mihailescu, M.R. & Bassell, G.J., hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA, Mol. Biol. Cell. 2016, 27(3):518-534. doi: 10.1091/mbc.E15-07-0504.
Mihailescu, R., Gene expression regulation: lessons from noncoding RNAs, RNA 2015, 21(4): 695-696. (invited article).
Stefanovic S., DeMarco, B.A., Underwood, A., Williams, K.R., Bassell, G.J. & Mihailescu, M.R., Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA, Mol Biosyst. 2015 11(12):3222-30. doi: 10.1039/c5mb00423c. (featured on the cover of the Molecular Biosystems Journal)
Stefanovic, S., Bassell G.J. & Mihailescu, M.R., G quadruplex RNA structures in PSD -95 mRNA: potential regulators of miR-125a seed binding site accessibility, RNA. 2015 21(1):48-60. doi: 10.1261/rna.046722.114.
Zhang, Y., Gaetano, C., Williams, K.R., Bassell G.J. & Mihailescu, M.R., FMRP interacts with G quadruplex structures in the 3'-UTR of its dendritic target Shank1 mRNA, RNA Biol. 2014;11(11):1364-74. doi: 10.1080/15476286.2014.996464.
Blice-Baum AC. & Mihailescu M.R., Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms. RNA. 2014, 20(1):103-14. Epub 2013 Nov 18.