We found that most miRNA genes, similar to protein coding genes, exhibited only two major modes of differential expression during differentiation and synaptogenesis in E17.5 mouse primary neuronal cultures. One set of miRNAs was continuously decreasing while another was continuously increasing in expression levels. Analysis of mRNA coding genes and miRNAs displaying these two trends showed that miRNAs were expressed inversely to their highly expressed predicted targets. This inverse relationship is consistent with previous results in other systems [12
Those miRNAs showing common expression profiles were found to cluster within genomic loci. In total, 47% of co-expressed miRNAs were localized within just five genomic clusters on chromosomes 12, 14 and X. It was previously shown that miRNA genes localized in the same genomic cluster can be co-transcribed, co-regulated, share evolutionary origin and have common functions [39
]. Thus it is likely that many co-expressed miRNAs in neuronal cultures have related functionality. The fact that a significantly high proportion of miRNAs, which are co-expressed in our study, have closely related seed regions supports this view.
Through a combination of mRNA and miRNA expression profiling and computational target prediction, we studied the regulatory potential of expressed miRNAs. It has previously been shown that miRNA regulation can be observed at both the mRNA [1
] and protein level [2
] and that there is reasonable agreement between the two.
Co-expression of genomically co-localized miRNAs and the apparent relatedness of their seed regions suggested using a combinatorial approach for functional analysis of predicted targets. The combined list of predicted targets of down-regulated miRNAs showed a significant enrichment in PSP genes. This implies that miRNAs developmentally down-regulated at earlier stages participate in the repression of a number of key neuronal genes at the time when neuronal network activity is undetectable [4
]. This is consistent with finding the enrichment of cytoplasmic localization, protein binding and transport activity GO terms among the targets of the down-regulated miRNAs. On the other hand, targets of up-regulated miRNAs are not enriched in the PSP genes. Additionally, nuclear localization, RNA splicing and chromatin modification GO terms are enriched among the targets of up-regulated miRNAs (see Additional file 8
for GO terms list). Thus it is possible that this category of targets was involved in the reprogramming of progenitor gene expression to a differentiated neuronal cell expression, which naturally occurs during the tested developmental time-window. This view is further supported by pathway/network enrichment analysis and suggests that miRNA regulation is important for the organization of large sets of the neuronal proteome at different stages of development.
In addition to differentially expressed miRNAs we have identified a third distinct type of miRNA expression which is the steady-state expression at relatively high levels. Many of the miRNAs that were previously linked to neuronal biology (e.g. mmu-miR-124, mmu-miR-125 family, mmu-miR-137, mmu-miR-128, mmu-miR-9 and mmu-let-7) [6
] belong to this category. Interestingly, we found that steady-state highly expressed miRNAs were predominantly located in isolated genomic loci rather than situated in genomic clusters of more than two miRNAs. We also found words complementary to the seed regions of at least three steady-state highly expressed miRNAs to rank among the most enriched words in 3'UTRs of relatively lowly expressed genes. Another 13 steady-state highly expressed miRNAs left a clear imprint on the global mRNA expression profile: A strong depletion signal for the seed-matching words of these miRNAs was observed in the 3'UTRs of highly expressed genes. These findings indicate that targets of many steady-state highly expressed miRNAs are likely to reside among relatively lowly expressed genes. Such categories of putative targets are likely to include many non-neuronal genes (e.g. immune system genes) and also genes involved in neuronal development at earlier timepoints (e.g. progenitor maintenance factors).