FMRP regulation of aggrecan mRNA translation controls perineuronal net development.

Perineuronal nets (PNNs) are mesh-like structures on the surfaces of parvalbumin-expressing inhibitory and other neurons, and consist of proteoglycans such as aggrecan, brevican, and neurocan. PNNs regulate the Excitatory/Inhibitory (E/I) balance in the brain and are formed at the closure of critical periods of plasticity during development. PNN formation is disrupted in Fragile X Syndrome, which is caused by silencing of the fragile X messenger ribonucleoprotein 1 (Fmr1) gene and loss of its protein product FMRP. FXS is characterized by impaired synaptic plasticity resulting in neuronal hyperexcitability and E/I imbalance. Here, we investigate how PNN formation is altered in FXS. PNNs are reduced in Fmr1 KO mouse brain when examined by staining for the lectin Wisteria floribunda agglutin (WFA) and aggrecan. Examination of PNNs by WFA staining at P14 and P42 in the hippocampus, somatosensory cortex, and retrosplenial cortex shows that they were reduced in these brain regions at P14 but mostly less so at P42 in Fmr1 KO mice. However, some differential FMRP regulation of PNN development in these brain regions persists, perhaps caused by asynchrony in PNN development between brain regions in wild-type animals. During development, aggrecan PNN levels in the brain were reduced in all brain regions in Fmr1 KO mice. Aggrecan mRNA levels were unchanged at these times, suggesting that FMRP is normally an activator of aggrecan mRNA translation. This hypothesis is buttressed by the observations that FMRP binds aggrecan mRNA and that ribosome profiling data show that aggrecan mRNA is associated with reduced numbers of ribosomes in Fmr1 KO mouse brain, indicating reduced translational efficiency. Moreover, aggrecan mRNA poly(A) tail length is also reduced in Fmr1 KO mouse brain, suggesting a relationship between polyadenylation and translational control. We propose a model where FMRP modulates PNN formation through translational up-regulation of aggrecan mRNA polyadenylation and translation.

FMRP deficiency leads to multifactorial dysregulation of splicing and mislocalization of MBNL1 to the cytoplasm.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that is often modeled in Fmr1 knockout mice where the RNA-binding protein FMRP is absent. Here, we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, PTBP1, and MBNL1. FMRP regulates the translation of Mbnl1 mRNA as well as Mbnl1 RNA auto-splicing. Elevated Mbnl1 auto-splicing in FMRP-deficient cells results in the loss of a nuclear localization signal (NLS)-containing exon. This in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This redistribution of MBNL1 isoforms in Fmr1-deficient cells could result in downstream splicing changes in other RNAs. Indeed, further investigation revealed that splicing disruptions resulting from Fmr1 depletion could be rescued by overexpression of nuclear MBNL1. Altered Mbnl1 auto-splicing also occurs in human FXS postmortem brain. These data suggest that FMRP-controlled translation and RNA processing may cascade into a general dys-regulation of splicing in Fmr1-deficient cells.

Antisense oligonucleotide rescue of CGG expansion-dependent FMR1 mis-splicing in fragile X syndrome restores FMRP.

Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1-expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion-dependent manner to generate the little-known FMR1-217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1-217 is also expressed in FXS premutation carrier-derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced FMR1, antisense oligonucleotide (ASO) treatment reduces FMR1-217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2'-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1-217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217, ASO treatment may offer a therapeutic approach to mitigate the disorder.

CPEB and translational control by cytoplasmic polyadenylation: impact on synaptic plasticity, learning, and memory.

The late 1990s were banner years in molecular neuroscience; seminal studies demonstrated that local protein synthesis, at or near synapses, was necessary for synaptic plasticity, the underlying cellular basis of learning and memory [1, 2]. The newly made proteins were proposed to "tag" the stimulated synapse, distinguishing it from naive synapses, thereby forming a cellular memory [3]. Subsequent studies demonstrated that the transport of mRNAs from soma to dendrite was linked with translational unmasking at synapses upon synaptic stimulation. It soon became apparent that one prevalent mechanism governing these events is cytoplasmic polyadenylation, and that among the proteins that control this process, CPEB, plays a central role in synaptic plasticity, and learning and memory. In vertebrates, CPEB is a family of four proteins, all of which regulate translation in the brain, that have partially overlapping functions, but also have unique characteristics and RNA binding properties that make them control different aspects of higher cognitive function. Biochemical analysis of the vertebrate CPEBs demonstrate them to respond to different signaling pathways whose output leads to specific cellular responses. In addition, the different CPEBs, when their functions go awry, result in pathophysiological phenotypes resembling specific human neurological disorders. In this essay, we review key aspects of the vertebrate CPEB proteins and cytoplasmic polyadenylation within the context of brain function.

CPEB1 regulates the inflammatory immune response, phagocytosis, and alternative polyadenylation in microglia.

Microglia are myeloid cells of the central nervous system that perform tasks essential for brain development, neural circuit homeostasis, and neural disease. Microglia react to inflammatory stimuli by upregulating inflammatory signaling through several different immune cell receptors such as the Toll-like receptor 4 (TLR4), which signals to several downstream effectors including transforming growth factor beta-activated kinase 1 (TAK1). Here, we show that TAK1 levels are regulated by CPEB1, a sequence-specific RNA binding protein that controls translation as well as RNA splicing and alternative poly(A) site selection in microglia. Lipopolysaccharide (LPS) binds the TLR4 receptor, which in CPEB1-deficient mice leads to elevated expression of ionized calcium binding adaptor molecule 1 (Iba1), a microglial protein that increases with inflammation, and increased levels of the cytokine IL6. This LPS-induced IL6 response is blocked by inhibitors of JNK, p38, ERK, NFκB, and TAK1. In contrast, phagocytosis, which is elevated in CPEB1-deficient microglia, is unaffected by LPS treatment or ERK inhibition, but is blocked by TAK1 inhibition. These data indicate that CPEB1 regulates microglial inflammatory responses and phagocytosis. RNA-seq indicates that these changes in inflammation and phagocytosis are accompanied by changes in RNA levels, splicing, and alternative poly(A) site selection. Thus, CPEB1 regulation of RNA expression plays a role in microglial function.

Oppositional poly(A) tail length regulation by FMRP and CPEB1.

Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3'UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases GLD2 (TENT2, PAPD4, Wispy) and GLD4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor GLD4 to RNA, we expressed double tagged GLD4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that coprecipitated with GLD4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA nanopore sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation.

Ribosome profiling reveals novel regulation of C9ORF72 GGGGCC repeat-containing RNA translation.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5' cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Do Fragile X Syndrome and Other Intellectual Disorders Converge at Aberrant Pre-mRNA Splicing?

Fragile X Syndrome is a neuro-developmental disorder caused by the silencing of the FMR1 gene, resulting in the loss of its protein product, FMRP. FMRP binds mRNA and represses general translation in the brain. Transcriptome analysis of the Fmr1-deficient mouse hippocampus reveals widespread dysregulation of alternative splicing of pre-mRNAs. Many of these aberrant splicing changes coincide with those found in post-mortem brain tissue from individuals with autism spectrum disorders (ASDs) as well as in mouse models of intellectual disability such as PTEN hamartoma syndrome (PHTS) and Rett Syndrome (RTT). These splicing changes could result from chromatin modifications (e.g., in FXS, RTT) and/or splicing factor alterations (e.g., PTEN, autism). Based on the identities of the RNAs that are mis-spliced in these disorders, it may be that they are at least partly responsible for some shared pathophysiological conditions. The convergence of splicing aberrations among these autism spectrum disorders might be crucial to understanding their underlying cognitive impairments.

The molecular biology of FMRP: new insights into fragile X syndrome.

Fragile X mental retardation protein (FMRP) is the product of the fragile X mental retardation 1 gene (FMR1), a gene that - when epigenetically inactivated by a triplet nucleotide repeat expansion - causes the neurodevelopmental disorder fragile X syndrome (FXS). FMRP is a widely expressed RNA-binding protein with activity that is essential for proper synaptic plasticity and architecture, aspects of neural function that are known to go awry in FXS. Although the neurophysiology of FXS has been described in remarkable detail, research focusing on the molecular biology of FMRP has only scratched the surface. For more than two decades, FMRP has been well established as a translational repressor; however, recent whole transcriptome and translatome analyses in mouse and human models of FXS have shown that FMRP is involved in the regulation of nearly all aspects of gene expression. The emerging mechanistic details of the mechanisms by which FMRP regulates gene expression may offer ways to design new therapies for FXS.

Noncanonical cytoplasmic poly(A) polymerases regulate RNA levels, alternative RNA processing, and synaptic plasticity but not hippocampal-dependent behaviours.

Noncanonical poly(A) polymerases are frequently tethered to mRNA 3' untranslated regions and regulate poly(A) tail length and resulting translation. In the brain, one such poly(A) polymerase is Gld2, which is anchored to mRNA by the RNA-binding protein CPEB1 to control local translation at postsynaptic regions. Depletion of CPEB1 or Gld2 from the mouse hippocampus results in a deficit in long-term potentiation (LTP), but only depletion of CPEB1 alters animal behaviour. To test whether a related enzyme, Gld4, compensates for the lack of Gld2, we separately or simultaneously depleted both proteins from hippocampal area CA1 and again found little change in animal behaviour, but observed a deficit in LTP as well as an increase in long-term depression (LTD), two forms of protein synthesis-dependent synaptic plasticity. RNA-seq data from Gld2, Gld4, and Gld2/Gld4-depleted hippocampus show widespread changes in steady state RNA levels, alternative splicing, and alternative poly(A) site selection. Many of the RNAs subject to these alterations encode proteins that mediate synaptic function, suggesting a molecular foundation for impaired synaptic plasticity.