A microRNA array reveals extensive regulation of microRNAs during brain development

Abstract

Several hundred microRNAs (miRNAs) have recently been cloned from a wide range of organisms across phylogeny. Despite the high degree of conservation of miRNAs, their functions in general, and in mammals particularly, are just beginning to be defined. Here we show that an oligonucleotide DNA array can be successfully used for the simultaneous analysis of miRNA expression profiles from tissues or cells. From a subset of miRNAs expressed in the brain we designed an oligonucleotide array spotted with probes specific for 44 mature miRNAs. These arrays demonstrated precise regulation of miRNA expression at mammalian brain developmental epochs. About 20% of the probed miRNAs changed significantly in their expression during normal brain development, and two of them, miR-9 and miR-131, were dysregulated in presenilin-1 null mice exhibiting severe brain developmental defects. Transcripts with regulated expression patterns on the arrays were validated by Northern blots. Additionally, a bioinformatic analysis of developmentally regulated miRNAs suggested potential mRNA targets. The arrays also revealed miRNAs distributed to translating polyribosomes in primary neurons where they are likely to modulate translation. Therefore, oligonucleotide arrays provide a new tool for studying miRNA expression in a variety of biological and pathobiological settings. Creating clusters of coexpressed miRNAs will contribute to understanding their regulation, functions, and discovery of mRNA targets.

FIGURE 1.

RNA samples enriched in LMW molecules were used as a probe to hybridize to oligo arrays. Total RNA was filtrated through Microcon YM-100 to obtain a fraction enriched in LMW RNA. Total RNA input (lanes 1,4), retaining fraction (lanes 2,5), and LMW RNA (lanes 3,6) were resolved into 15% TBE-Urea RNA gels and stained with Ethidium Bromide (lanes 1–3) or analyzed by Northern blotting with a probe specific for miR-124a (lanes 4–6). In addition to miR-124a, its precursor is detected in lanes 4 and 5, but not in a lane 6. Synthetic RNA markers of indicated size were added to an input total RNA sample. tRNAs here comigrate with a synthetic marker of 65 nt.

FIGURE 2.

Oligonucleotide arrays reveal differential expression of miRNAs during corticogenesis. (A) Representative oligonucleotide arrays on which ³³P-labeled LMW RNA samples from E12 and E13 rat forebrains were hybridized. The arrays were analyzed to predict differentially regulated miRNAs. (B) Expression ratios of differentially expressed miRNA as calculated from arrays. RNA at developmental stages E12, E13, E21, and adult were hybridized to arrays and analyzed. Expression ratios are shown only where the ratio exceeds twofold and the corresponding p-value is equal or below 0.05. (C) Northern blot hybridizations of total RNA confirm regulated expression patterns of nine miRNAs during brain prenatal (E) and postnatal (P) development. 5S rRNA was detected by ethidium bromide staining of the gels prior to transfer to verify equal loading of total RNA.

FIGURE 3.

Characterization of developmentally regulated miR-9 and miR-131 transcripts. (A) Northern blots of total RNA isolated from cerebral cortex (co), cerebellum (ce), kidney (ki), heart (ht), liver (li), lungs (lg), ovary (ov), thymus (thy), and testis (te), probed with miR-9 and mir-131. (B) Predicted secondary structures for putative precursors of multicopy human and rodent miR-9 and miR-131 cotranscribed pair. RNA secondary structure prediction was performed using mfold software. MiR-9 and miR-131 sequences are shown in bold. (C) Northern blots of total RNA isolated from pre- and postnatal developing brain with probes specific for the loop region of the predicted mir-9/miR-131 precursors. Oligonucleotides used as probes were 5′-TTATGAAGACTCCA CACCAC-3′ for chromosome 1 precursor, 5′-TTTATGAAGACCAATACAC-3′ for chromosome 5 precursor, and 5′-TTATGACGGCTCTGTGGCAC-3′ for chromosome 15 precursor. The putative precursor encoded on chromosome 5 was undetectable.

FIGURE 4.

MiR-9 and miR-131 are disregulated in PS1 null mice. Northern blots of total brain RNA isolated from PS1(+/−) and PS1(−/−) embryos of indicated ages (two embryos per each group), probed for miR-9 and miR-131. PS1(+/−) heterozygous are phenotypically undistinguishable from wild-type animals. Bands were quantified and represented as bars in the lower panel.

FIGURE 5.

miRNAs are differentially associated with translating polyribosomes. (A) Representative oligo arrays showing hybridization with ³³P-labeled LMW RNA that was isolated from mRNP and polysomal fractions of cultured primary neurons. Fifty percent of the RNA from the sucrose gradient fractions was used. Signals corresponding to miR-9 and miR-131 hybridizations are indicated. (B) Oligonucleotide arrays predict differential association of miR-9 and miR-131 with polyribosomes. mRNP and polysomal RNAs were isolated from three independent fractionation experiments, and each LMW RNA sample was hybridized in triplicate to the array membranes as shown in A. MiR-9 and miR-131 spots were quantified and the ratio of miR-9 to miR-131 signals was calculated for filters hybridized with polysomal RNA versus mRNP’s RNA. The bars represent an average of three completely independent experiments. (C) Northern blot hybridization of RNA isolated from sucrose gradient fractions with miR-9 and miR-131 specific probes. Fifty percent of RNA from the fraction was loaded per lane. Bands were quantified by densitometry and plotted, confirming that higher proportion of miR-9 than of miR-131 is associated with actively translating polysomes.

FIGURE 6.

Putative mRNA targets. Sequence alignments for neuronal developmentally regulated miRNAs and their putative human and mouse mRNA targets. Black boxes indicate sequences conserved between human and mice. DataBank accession numbers for mRNAs are indicated.