Abstract
Patterning of the animal embryo's antero-posterior (AP) axis is dependent on spatially and temporally regulated Hox gene expression. The murine Hoxd4 gene has been proposed to harbour two promoters, an upstream promoter P2, and a downstream promoter P1, that lie 5.2 and 1.1 kilobase pairs (kb) upstream of the coding region respectively. The evolutionarily conserved microRNA-10b (miR-10b) gene lies in the Hoxd4 genomic locus in the intron separating the non-coding exons 4 and 5 of the P2 transcript and directly adjacent to the proposed P1 promoter. Hoxd4 transcription is regulated by a 3′ neural enhancer that harbours a retinoic acid response element (RARE). Here, we show that the expression profiles of Hoxd4 and miR-10b transcripts during neural differentiation of mouse embryonal carcinoma (EC) P19 cells are co-ordinately regulated, suggesting that both Hoxd4 and miR-10b expression is governed by the neural enhancer. Our observation that P1 transcripts are uncapped, together with the mapping of their 5′ ends, strongly suggests that they are generated by Drosha cleavage of P2 transcripts rather than by transcriptional initiation. This is supported by the colocalization of P1 and P2 transcripts to the same posterior expression domain in the mouse embryo. These uncapped P1 transcripts do not appear to possess an Internal Ribosomal Entry Site (IRES), but accumulate within multiple punctate bodies within the nucleus suggesting that they play a functional role. Finally, similar uncapped Drosha-cleaved P1-like transcripts originating from the paralogous Hoxb4/miR-10a locus were also identified. We propose that these transcripts may belong to a novel class of regulatory RNAs.
Highlights
MicroRNAs are a class of highly conserved small noncoding RNAs expressed in a wide range of organisms [1,2]
To determine if the expression of pri-miR-10b is controlled by the Hoxd4 neural enhancer, the relative levels of Hoxd4 P1, P2 and miR10b transcripts were measured in differentiating P19 mouse embryonal carcinoma (EC) cells
This delay in the expression peak may reflect differences in processing or stability; the overall expression profile is similar. This is in contrast to the expression profiles of other Hox genes such as Hoxa1 which is induced by 6 h and peaks as early as 48 h of neural differentiation [37,39]
Summary
MicroRNAs are a class of highly conserved small noncoding RNAs (ncRNAs) expressed in a wide range of organisms [1,2]. The pri-miRNA forms a hairpin-loop structure that is cleaved at its base by an RNAse III enzyme, Drosha, to form the precursor microRNA (pre-miRNA) which is exported out of the nucleus and cleaved again on the loop side of the hairpin by another RNAse III enzyme, Dicer This generates an miRNA: miRNA* duplex, one strand of which is preferentially selected and incorporated into the RNA-induced silencing complex (RISC). The single stranded mature miRNA is typically 21–23 nucleotides long and functions by base-pairing to target complementary mRNAs to regulate gene expression In animals, this regulation occurs mostly, but not always, at the post-transcriptional level [3]. Recent evidence suggests that miRNAs are able to epigenetically silence genes at the transcriptional level [4]
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