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Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells.

Peschansky VJ, Pastori C, Zeier Z, Motti D, Wentzel K, Velmeshev D, Magistri M, Bixby JL, Lemmon VP, Silva JP, Wahlestedt C - Front Genet (2015)

Bottom Line: Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays.Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4.Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

View Article: PubMed Central - PubMed

Affiliation: Center for Therapeutic Innovation and Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami Miami, FL, USA.

ABSTRACT
CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. "Full"-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4's function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

No MeSH data available.


Related in: MedlinePlus

Expression of FMR4 and its target MBD4 during differentiation of hNPCs may be related to chromatin-modulatory function of the lncRNA. (A) Comparison of RNA expression between proliferating neurospheres (0 DIV) and differentiating hNPCs (5 DIV) revealed increased FMR1 and decreased FMR4 after 5 days of differentiation (n = 5, *p < 0.05 by Student’s t-test). As predicted, the putative FMR4 target gene MBD4 was upregulated with decreased FMR4. Subcellular fractionation of hNPCs with subsequent qPCR detection confirmed the cytoplasmic localization of mRNAs such as (B)GAPDH and FMR1. MALAT1(C), known to be associated to nuclear speckles, was significantly enriched in the nucleus and chromatin compared to cytoplasm. FMR4 RNA (C) was largely localized to chromatin. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANOVA with Tukey HSD).
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Figure 2: Expression of FMR4 and its target MBD4 during differentiation of hNPCs may be related to chromatin-modulatory function of the lncRNA. (A) Comparison of RNA expression between proliferating neurospheres (0 DIV) and differentiating hNPCs (5 DIV) revealed increased FMR1 and decreased FMR4 after 5 days of differentiation (n = 5, *p < 0.05 by Student’s t-test). As predicted, the putative FMR4 target gene MBD4 was upregulated with decreased FMR4. Subcellular fractionation of hNPCs with subsequent qPCR detection confirmed the cytoplasmic localization of mRNAs such as (B)GAPDH and FMR1. MALAT1(C), known to be associated to nuclear speckles, was significantly enriched in the nucleus and chromatin compared to cytoplasm. FMR4 RNA (C) was largely localized to chromatin. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANOVA with Tukey HSD).

Mentions: The FMR1 locus is crucial to normal brain development; thus, we wanted to determine whether FMR4 expression is dependent on developmental stage. We extracted RNA from undissociated hNS [“0 days in vitro” (DIV)] and cultured cells from dissociated hNS up to five DIV in differentiation media. At both time points, we measured FMR4 expression as well as that of FMR1, to determine whether these transcripts are independently regulated. We observed that FMR4 expression is significantly decreased in differentiating cells at 5 DIV while FMR1 is increased at the same time point (Figure 2A). We then measured MBD4 and found that it is also developmentally regulated. As indicated by the microarray analysis, MBD4 was increased at a time point when FMR4 expression is low (Figure 2A). We also observed changes in expression level of other FMR4 target genes in undifferentiated, proliferating hNPCs transduced with an mCherry-tagged lentiviral vector expressing FMR4 (Supplementary Figure S2A). We found that FMR4 overexpression significantly upregulated two putative targets, the deubiquitinase YOD1 and the G-protein subunit GNG12, and downregulated the ribonucleotide reductase RRM2 (Supplementary Figure S2B). These data corroborate our finding that FMR4 regulates gene expression in trans.


Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells.

Peschansky VJ, Pastori C, Zeier Z, Motti D, Wentzel K, Velmeshev D, Magistri M, Bixby JL, Lemmon VP, Silva JP, Wahlestedt C - Front Genet (2015)

Expression of FMR4 and its target MBD4 during differentiation of hNPCs may be related to chromatin-modulatory function of the lncRNA. (A) Comparison of RNA expression between proliferating neurospheres (0 DIV) and differentiating hNPCs (5 DIV) revealed increased FMR1 and decreased FMR4 after 5 days of differentiation (n = 5, *p < 0.05 by Student’s t-test). As predicted, the putative FMR4 target gene MBD4 was upregulated with decreased FMR4. Subcellular fractionation of hNPCs with subsequent qPCR detection confirmed the cytoplasmic localization of mRNAs such as (B)GAPDH and FMR1. MALAT1(C), known to be associated to nuclear speckles, was significantly enriched in the nucleus and chromatin compared to cytoplasm. FMR4 RNA (C) was largely localized to chromatin. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANOVA with Tukey HSD).
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Expression of FMR4 and its target MBD4 during differentiation of hNPCs may be related to chromatin-modulatory function of the lncRNA. (A) Comparison of RNA expression between proliferating neurospheres (0 DIV) and differentiating hNPCs (5 DIV) revealed increased FMR1 and decreased FMR4 after 5 days of differentiation (n = 5, *p < 0.05 by Student’s t-test). As predicted, the putative FMR4 target gene MBD4 was upregulated with decreased FMR4. Subcellular fractionation of hNPCs with subsequent qPCR detection confirmed the cytoplasmic localization of mRNAs such as (B)GAPDH and FMR1. MALAT1(C), known to be associated to nuclear speckles, was significantly enriched in the nucleus and chromatin compared to cytoplasm. FMR4 RNA (C) was largely localized to chromatin. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 by One-way ANOVA with Tukey HSD).
Mentions: The FMR1 locus is crucial to normal brain development; thus, we wanted to determine whether FMR4 expression is dependent on developmental stage. We extracted RNA from undissociated hNS [“0 days in vitro” (DIV)] and cultured cells from dissociated hNS up to five DIV in differentiation media. At both time points, we measured FMR4 expression as well as that of FMR1, to determine whether these transcripts are independently regulated. We observed that FMR4 expression is significantly decreased in differentiating cells at 5 DIV while FMR1 is increased at the same time point (Figure 2A). We then measured MBD4 and found that it is also developmentally regulated. As indicated by the microarray analysis, MBD4 was increased at a time point when FMR4 expression is low (Figure 2A). We also observed changes in expression level of other FMR4 target genes in undifferentiated, proliferating hNPCs transduced with an mCherry-tagged lentiviral vector expressing FMR4 (Supplementary Figure S2A). We found that FMR4 overexpression significantly upregulated two putative targets, the deubiquitinase YOD1 and the G-protein subunit GNG12, and downregulated the ribonucleotide reductase RRM2 (Supplementary Figure S2B). These data corroborate our finding that FMR4 regulates gene expression in trans.

Bottom Line: Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays.Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4.Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

View Article: PubMed Central - PubMed

Affiliation: Center for Therapeutic Innovation and Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of Miami Miami, FL, USA.

ABSTRACT
CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. "Full"-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis-regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4-responsive genes, including the methyl-CpG-binding domain protein 4 (MBD4). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4) and MBD4. We therefore propose that FMR4's function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

No MeSH data available.


Related in: MedlinePlus