abd-A Regulation by the iab-8 Noncoding RNA

The homeotic genes in Drosophila melanogaster are aligned on the chromosome in the order of the body segments that they affect. The genes affecting the more posterior segments repress the more anterior genes. This posterior dominance rule must be qualified in the case of abdominal-A (abd-A) repression by Abdominal-B (Abd-B). Animals lacking Abd-B show ectopic expression of abd-A in the epidermis of the eighth abdominal segment, but not in the central nervous system. Repression in these neuronal cells is accomplished by a 92 kb noncoding RNA. This “iab-8 RNA” produces a micro RNA to repress abd-A, but also has a second, redundant repression mechanism that acts only “in cis.” Transcriptional interference with the abd-A promoter is the most likely mechanism

ABD-A expression in <i>Abd-B</i> mutant embryos.

<p>Stage 14 embryos in A–C were stained with antibody to ABD-A, opened along the dorsal midline and flattened for photography. ABD-A is absent from PS13 in wild type (A), but appears throughout PS13 in <i>Df(3R)C4</i> homozygotes (B). In <i>Abd-B^D16</i> homozygotes, ABD-A is only in the lateral and dorsal epidermis of PS13 (C). Dissected CNS's from stage15 embryos in D were doubly stained for ABD-A (red) and ABD-B (green). In wild type, the expression domains overlap through PS10-12, with some nuclei expressing both proteins. In <i>Abd-B^D14</i> homozygotes, ABD-B expression is absent from PS10-13, but the ABD-A pattern is unchanged, leaving PS13 without either protein.</p

Evolutionary conservation.

<p>A. A comparison of exon 3 and neighboring bases with the homologous regions from the genomes of three other <i>Drosophila</i> species. B. Potential nine amino acid peptide within exon 8 of the iab-8 ncRNA. The <i>D. melanogaster</i> sequence is compared to that of <i>D. ananassae</i>. The initial methionine codon is preceded by a perfect translation start consensus sequence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Cavener1" target="_blank">[46]</a>, and there are two stop codons after the 9th amino acid. The three bases altered in <i>D. ananassae</i> are highlighted in red; only one changes the predicted amino acid.</p

Map of the abdominal half of the bithorax complex.

<p>The horizontal bar indicates the DNA sequence map, numbered in kb according to Martin et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Martin1" target="_blank">[4]</a> (Genbank U31961). Base #1 corresponds to base 12,809,162 on chromosome 3R in release 5.37 of the <i>Drosophila</i> genome. The coordinates proceed distal to proximal on chromosome 3R, which is opposite in orientation to the whole genome numbering. The regulatory domains <i>iab-2</i> through <i>iab-8</i> are color coded; the domain borders are defined by deletion mutations (<i>Fab8 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Barges1" target="_blank">[<i>22</i>]</a>, ; <i>Fab7</i>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Gyurkovics1" target="_blank">[41]</a>; <i>Mcp</i>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Karch3" target="_blank">[44]</a>; <i>iab-3/iab-4</i>, L. Sipos personal communication), or inferred from the binding sites of the CTCF factor <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002720#pgen.1002720-Holohan1" target="_blank">[45]</a>. Below the DNA bar are shown the splicing patterns of <i>abd-A</i> and <i>Abd-B</i> (in black), a cDNA derived from the <i>Mi(Hto-WP)LNP</i> insertion (red), and the MIP06894 cDNA (green). At the bottom, the splicing pattern for the iab-8 ncRNA is shown in dark blue, with numbered exons, and alternate 5′ or 3′ extensions indicated with light blue lines. Mutant lesions are indicated above the DNA bar. The rearrangement breakpoints are color coded according to their phenotypes when heterozygous with the <i>mfs5649</i> insertion.</p

ABD-A expression in rearrangements truncating the iab-8 ncRNA.

<p>Embryos homozygous for the indicated mutations were doubly stained for ENGRAILED (green) and ABD-A (red), and the CNS's were dissected and photographed. The posterior end of each CNS is shown; the ENGRAILED stripes mark the parasegmental boundaries. The <i>iab-4</i> and <i>iab-7</i> breaks cause widespread misexpression of ABD-A in PS13, but <i>iab-3</i> breaks show only subtle misexpression in a few nuclei. Embryos homozygous for a deletion of the iab-8 miRNA also show misexpression in only a few nuclei.</p