FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation

Abstract

SummaryFMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2’O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2’O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns

“…The human embryonic stem cell (hESC) line Shef 4, hereafter referred to as FMR1 +/y , was obtained from the UK Stem Cell Bank [26]. The Shef 4 FMR1 null line, hereafter referred to as FMR1 −/y , was generated using CRISPR-Cas9 technology as described previously [24]. hiPSC and hESC colonies were cultured and propagated in xeno-free and feeder-free conditions using Essential 8 medium (Thermo Fisher Scientific) in 6-well plates (Nunc Nunclon delta surface, Thermo Fisher Scientific) coated with reduced growth-factor Matrigel (Corning Inc, New York, NY) at 37°C, 5% CO 2 in a humidified incubator.…”

“…Fibroblast-derived iPSCs were generated from two healthy individuals (CON1 and CON2), three FXS patients lacking FMRP (FXS1, FXS2 and FXS3) and one isogenic embryonic stem cell (FMR1 +/y ; FMR1 −/y ) pair where the FMR1 gene was deleted using CRISPR/Cas9-mediated genome editing [24]. The absence of FMRP in FXS1, FXS2, FXS3 and FMR1 −/y PSC colonies was confirmed using western blot analysis as shown in Fig.…”

“…Given the large number of FMRP targets that are associated with synaptic function and the well-described synaptic and network properties that are dysregulated in pre-clinical models of FXS, our present study focuses on assessing the physiological properties of two control (CON1, CON2) and three FXS patient lines (FXS1, FXS2, FXS3) of human cortical neurons derived from induced pluripotent stem cells (iPSCs). In addition, to ensure that the differences we observe in FXS patient lines are due to the silencing of the FMR1 gene and the absence of FMRP and not differences in the different genetic backgrounds or 'environmental' influences on the iPSC-derived neurons, we also describe the properties of an isogenic pair of lines [24], one of which has had the FMR1 gene genetically deleted (FMR1 −/y ) to allow direct comparison with an otherwise genetically identical line (FMR1 +/y ). Using electrophysiological recordings, we report that there is altered spontaneous neuronal action potential burst firing activity in all lines that lack FMRP.…”

Ribosomes: An Exciting Avenue in Stem Cell Research

“…FMRP has been shown to function predominantly in the cytoplasm; however, recent evidence has demonstrated its role in the nucleus [ 98 , 99 ]. In the nuclei of human embryonic stem cells, FMRP directly interacts with C/D box snoRNAs and results in the 2′-O-Me modification of rRNA, thereby causing ribosome heterogeneity by affecting rRNA folding and ribosomal assembly [ 100 , 101 ]. In the cytoplasm, FMRP identifies 2′-O-Me-modified ribosomes to enable specific translation of its target mRNAs [ 101 , 102 ].…”

“…In the nuclei of human embryonic stem cells, FMRP directly interacts with C/D box snoRNAs and results in the 2′-O-Me modification of rRNA, thereby causing ribosome heterogeneity by affecting rRNA folding and ribosomal assembly [ 100 , 101 ]. In the cytoplasm, FMRP identifies 2′-O-Me-modified ribosomes to enable specific translation of its target mRNAs [ 101 , 102 ]. FMRP promotes the expression of genes involved in stem cell intracellular pathways, such as mTOR, PI3K, ERK, and Gsk3 β [ 103 – 106 ].…”