Antisense oligonucleotide rescue of CGG expansion–dependent
            <i>FMR1</i>
            mis-splicing in fragile X syndrome restores FMRP

Shah, Sneha; Sharp, Kevin J.; Ponny, Sithara Raju; Watts, Jonathan K.; Berry‐Kravis, Elizabeth; Richter, Joel D.

doi:10.1073/pnas.2302534120

Cited by 18 publications

(4 citation statements)

References 45 publications

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“…Gene expression and RNA splicing are mis-regulated in the Fmr1 -deficient mouse hippocampus [ 15 ] and FXS patient-derived blood samples [ 26 ]. To determine whether this mis-regulation occurs in other brain regions and in peripheral tissues from mice, we sequenced RNA from ( n = 3, 2- to 3-month-old) WT and Fmr1 KO hippocampus, cerebellum, and cortex, as well as liver, muscle, and testis ( Fig 1A ).…”

Section: Resultsmentioning

confidence: 99%

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

Jung,

Shah,

Han

et al. 2023

PLoS Biol

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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.

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Section: Resultsmentioning

confidence: 99%

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

Jung,

Shah,

Han

et al. 2023

PLoS Biol

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“…A number of strategies have been developed to reduce the symptoms of FXS through pharmacology (Davenport et al, 2016; Yamasue et al, 2019), reactivating FMRP expression (Xie et al, 2016; Graef et al, 2019; Yrigollen and Davidson, 2019; Shah et al, 2023), or inducing FMRP expression through delivery of viral constructs (Zeier et al, 2009; Gholizadeh et al, 2014; Hampson et al, 2019; Wong et al, 2023). An early study attempted to reintroduce the full length FMRP conjugated to tat as a cell permeable protein, but reported toxicity when tested in fibroblasts (Reis et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…A number of strategies have been developed to reduce the symptoms of FXS through pharmacology (Yamasue et al, 2019), reactivating FMRP expression (Xie et al, 2016;Graef et al, 2019;Yrigollen and Davidson, 2019;Shah et al, 2023), or inducing FMRP expression through delivery of viral constructs (Hampson et al, 2019). It was recently found that reintroducing the N-terminus of FMRP as a tat conjugate peptide achieved rapid transport across the BBB, rescuing circuit functions and alleviating symptoms of Fmr1 KO mice (Zhan et al, 2020).…”

Section: Rescue Of Fmrp Function In Fxsmentioning

confidence: 99%

Calcium-dependent regulation of neuronal excitability is rescued in Fragile X Syndrome by a tat-conjugated N-terminal fragment of FMRP

Zhan,

Asmara,

Pfaffinger

et al. 2024

Preprint

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Fragile X Syndrome arises from the loss of Fragile X Messenger Ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber LTP by adjusting Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knockout (KO) tsA-201 cells and cerebellar sections from Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex, and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signalling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signalling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of Fragile X Syndrome.

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“…(CAG)n NRE in huntingtin (HTT) exon 1 caused aberrant splicing and pre-mature cleavage and polyadenylation in both knock-in mouse models of HD and patient tissues 48 , resulting in truncated yet still translatable mRNAs for polyQ synthesis. In addition to transcriptional silencing, mis-splicing of FMR1 mRNAs in individuals with fragile X syndrome further reduces FMRP abundance 49 . Furthermore, multiple intronic NREs have been shown to suppress canonical splicing and to cause the retention of their respective host introns 50,51 , including (CCTG)n in CNBP intron 1 (DM2), (CTG)n in TCF4 intron 3 (Fuchs endothelial corneal dystrophy), and (GGCCTG)n. in NOP56 intron 1 (SCA36).…”

Section: Discussionmentioning

confidence: 99%

Aberrant splicing exonizesC9ORF72repeat expansion in ALS/FTD

Yang,

Wijegunawardana,

Sheth

et al. 2023

Preprint

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A nucleotide repeat expansion (NRE) in the first annotated intron of theC9ORF72gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While C9 NRE-containing RNAs can be translated into several toxic dipeptide repeat proteins, how an intronic NRE can assess the translation machinery in the cytoplasm remains unclear. By capturing and sequencing NRE-containing RNAs from patient-derived cells, we found that C9 NRE was exonized by the usage of downstream 5ʹ splice sites and exported from the nucleus in a variety of spliced mRNA isoforms.C9ORF72aberrant splicing was substantially elevated in both C9 NRE+motor neurons and human brain tissues. Furthermore, NREs above the pathological threshold were sufficient to activate cryptic splice sites in reporter mRNAs. In summary, our results revealed a crucial and potentially widespread role of repeat-induced aberrant splicing in the biogenesis, localization, and translation of NRE-containing RNAs.

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

Cited by 18 publications

References 45 publications

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

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

Calcium-dependent regulation of neuronal excitability is rescued in Fragile X Syndrome by a tat-conjugated N-terminal fragment of FMRP

Aberrant splicing exonizesC9ORF72repeat expansion in ALS/FTD

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