Knockdown and Overexpression of FMR4 in HEK293T cells at 6 and 24 hours. Homo sapiens

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, FXTAS). However, the FMR1 locus is complex and encodes several long noncoding RNAs (lncRNAs), 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, and further investigated their function related to human neural precursor cells. 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. Overall design: Study design includes 2 timepoints (6 and 24 hrs post-transfection) x 4 conditions (knockdown & control, overexpression & control) x 3 biological replicates. 3 technical replicates were pooled to creat each biological replicate.

脆性X智力低下1号（Fragile X mental retardation 1, FMR1）基因中的CGG重复扩增可引发一系列相关疾病，此类疾病的临床表现为智力障碍与自闭症（即脆性X综合征，Fragile X Syndrome, FXS），或成年起病的神经退行性病变（即脆性X相关震颤/共济失调综合征，Fragile X-associated Tremor/Ataxia Syndrome, FXTAS）。然而，FMR1基因座结构复杂，可编码多种长链非编码RNA（long noncoding RNA, lncRNA），其表达会因重复扩增突变发生改变。目前这类lncRNA的功能尚未明确，因此我们对此前已鉴定的FMR4展开了功能研究。FMR1三联体重复的“全长”扩增会同时沉默FMR1与FMR4的表达，因此我们重点关注可能加重FXS表型表现的功能丧失效应。由于这两种转录本不存在相互顺式调控，我们借助基因表达微阵列，探究了FMR4对远端基因组位点靶基因的调控潜能。我们成功鉴定出FMR4响应基因，并进一步研究了这些基因在人神经前体细胞中的相关功能。据此我们提出，FMR4作为基因调控型lncRNA发挥功能，且该转录本可能参与正常发育过程。对FMR4的深入研究，有助于我们增进对调控型lncRNA的作用以及FMR1重复扩增后果的理解。整体实验设计：本研究设计包含2个时间点（转染后6小时与24小时）×4种实验条件（敲低组与对照组、过表达组与对照组）×3次生物学重复。每个生物学重复由3次技术重复混合制备而成。