MicroRNAs in the Drosophila bithorax complex

Abstract

The iab-4 noncoding RNA from the Drosophila bithorax complex is the substrate for a microRNA (miRNA). Gene conversion was used to delete the hairpin precursor of this miRNA; flies homozygous for this deletion are sterile. Surprisingly, this mutation complements with rearrangement breakpoint mutations that disrupt the iab-4 RNA but fails to complement with breaks mapping in the iab-5 through iab-7 regulatory regions. These breaks disrupt the iab-8 RNA, transcribed from the opposite strand. This iab-8 RNA also encodes a miRNA, detected on Northern blots, derived from the hairpin complementary to the iab-4 precursor hairpin. Ultrabithorax is a target of both miRNAs, although its repression is subtle in both cases.

Figure 1.

The abdominal half of the BX-C, showing iab-4 and iab-8 ncRNAs, and the rearrangement breakpoints tested for complementation. The horizontal black line marks the sequence coordinates, following the convention of Martin et al. (1995). The 10-kb region covering the iab-4 ncRNA transcription unit is expanded above. The iab-4 transcription unit is diagrammed in orange, and embryos stained for the iab-4 RNA are in the orange box (at top right). The iab-8 transcription unit is diagrammed with the long blue arrow in the bottom half of the figure. Probes B, 8E, and BPP were used to detect the iab-8 RNA in prior studies (see Results and Discussion). Embryos stained for the iab-8 RNA are in the blue box (at bottom left). Embryos are shown at stages 5 or 6 (for iab-4 or iab-8 RNAs), stage 10, stage 13, and stage 15 (Campos-Ortega and Hartenstein 1985). The 4.2-kb fragment used for making both strand-specific probes is shown at the top. Vertical arrows indicate the positions of rearrangement breaks relative to the maps; the horizontal bars on these arrows indicate the region of uncertainty for each break. Breakpoint chromosomes that gave sterile flies when heterozygous with the ΔmiRNA conversion chromosome are shown with red arrows. Breakpoints that were fertile are shown in green. Note that four breakpoints upstream of the miRNA hairpin in the iab-4 RNA are fertile; five breakpoints upstream of the hairpin in the iab-8 RNA are sterile. Two breakpoints closely flanking the site of the miRNA hairpin gave distinctive results, as indicated in pink and blue.

Figure 2.

DNA sequences of the wild-type miR-iab-4 precursor region and of the ΔmiRNA derivative. The black boxes on the top strand indicate the miR-iab-4 5p and miR-iab-4 3p sequences reported by Aravin et al. (2003). The gray boxes on the bottom strand indicate the hypothetical sequences of miR-iab-8 5p and miR-iab-8 3p, assuming they are near homologs to the known miRNAs from the top strand. The actual miR-iab-8 5p sequence is slightly shifted from this prediction (Stark et al. 2008), and miR-iab-8 3p has not been detected to date. Dots between the top and bottom strands mark the base pairs conserved in the mosquito. The BstZ17I restriction sites were used to replace most of the hairpin region with sequences for the HindIII and I-SceI restriction enzymes.

Figure 3.

Developmental Northern blots. The probe for the top panel was directed against miR-iab-4 5p and for the bottom panel, against miR-iab-8 5p. Total RNA was extracted from wild-type (Canton S) animals at the indicated stages and separated on an 11% denaturing polyacrylamide gel. The migration positions of Drosophila tRNA and 2S RNA are indicated.

Figure 4.

UBX protein expression in the CNS of stage 15 embryos (∼12 h old). The brackets to the left of each nerve chord mark the region of the third abdominal segment (PS8), and the brackets to the right mark the eighth abdominal segment (PS13). The miRNA⁻ embryo, lacking both miR-iab-4 and miR-iab-8 (ΔmiRNA homozygous), shows more uniform staining for UBX in the third through seventh abdominal segments, and ectopic staining in a few cells of the eighth abdominal segment. The repression of Ubx by ABD-A is much more dramatic, as illustrated in the abd-A⁻ homozygote (using the abd-A^D100.24 point mutation). Likewise, an Abd-B⁻ homozygote (the HCJ199ry⁻ mutation, a P-element insertion in the first exon of the Type I Abd-B mRNA) derepresses Ubx in the eighth abdominal segment much more dramatically than does miR-iab-8.