Trisomy-21 gene dosage over-expression of miRNAs results in the haploinsufficiency of specific target proteins

Abstract

Down syndrome (DS) or Trisomy 21 (Ts21) is caused by the presence of an extra copy of all or part of human chromosome 21 (Hsa21) and is the most frequent survivable congenital chromosomal abnormality. Bioinformatic annotation has established that Hsa21 harbors more than 400 genes, including five microRNA (miRNA) genes (miR-99a, let-7c, miR-125b-2, miR-155, and miR-802). MiRNAs are endogenous, single-stranded, small non-coding RNA molecules that regulate gene expression by interacting with specific recognition elements harbored within the 3'-untranslated region (3'-UTR) of mRNAs and subsequently target these mRNAs for translational repression or destabilization. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments have demonstrated that Hsa21-derived miRNAs were over-expressed in fetal brain and heart specimens isolated from individuals with DS. We now propose that Ts21 gene dosage over-expression of Hsa21-derived miRNAs in DS individuals result in the decreased expression of specific target proteins (i.e. haploinsufficiency) that contribute, in part, to the DS phenotype.

Figure 1

Relative expression of Hsa21-derived miRNAs in human tissues. Mature human let-7c, miR-99a, miR-125b, miR-155 and miR-802 were quantified utilizing TaqMan microRNA assay kits specific for each Hsa21-derived miRNA (Applied Biosystems, Foster City, CA) as previously described., The expression values were normalized to RNU48 for each tissue. Relative gene expression was calculated as 2^{-(CTmiR-155-CT-RNU48)}. The relative abundance of each Hsa-21 derived miRNA is shown as a percentage of total combined expression of Hsa-21 derived miRNAs for each tissue investigated.

Figure 2

Hsa21-derived miRNAs are overexpressed in human DS pre-frontal cortex brain specimens. Mature Hsa21-derived miRNAs were quantified utilizing RT-PCR as previously described by our laboratory,,, using total RNA isolated from human fetal (18–22 weeks of gestation), child (1–8 years), adolescent (9–19 years old) and adult (20–50 years old) prefrontal cortex specimens from control and DS (age- and sex-matched, n = 3–5) samples. Gene expression was calculated relative to 18S rRNA and the data are expressed as percent over control (variation between control samples was never greater than 5%), which was assigned a value of 100%. The error bars represent the average ± S.E. of triplicate samples repeated in at least three independent experiments (*p < 0.01 DS vs. control).

Figure 3

The human AT₁R +1166 A/C SNP occurs in the miR-155-binding site. (A) Complementarity between miR-155 and the hAT₁R 3′-UTR site targeted (70–90 bp downstream from the human AT₁R stop codon). The +1166 A/C SNP corresponds to the nucleotide 86 bp downstream from the human AT₁R stop codon (shown in bold print). The binding of miR-155 to the hAT₁R 3′-UTR target site fulfills the requirement of a 7 bp seed sequence of complementarity at the miRNA 5′ end when the +1166 A-allele is expressed. (B) Complementarity between miR-155 and the human AT₁R 3′-UTR harboring the +1166 C-allele. If the +1166 C-allele is expressed, the seed sequence requirement would not be met and, as a consequence, it would be expected that human AT₁R expression would be elevated.