The expression of Drosophila melanogaster Hox gene Ultrabithorax is not overtly regulated by the intronic long noncoding RNA lncRNA:PS4 in a wild-type genetic background

Abstract

Long non-coding RNAs (lncRNAs) have been implicated in a variety of processes in development, differentiation, and disease. In Drosophila melanogaster, the bithorax Hox cluster (BX-C) contains three Hox genes (Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)), along with a number of lncRNAs, most with unknown functions. Here, we investigated the function of a long non-coding RNA, lncRNA: PS4 that originates in the second intron of Ubx and is transcribed in the antisense orientation to Ubx. The expression pattern of lncRNA: PS4 is complementary to Ubx in the thoracic primordia, and the lncRNA: PS4 coding region overlaps the location of the large insertion that causes the dominant homeotic mutation Contrabithorax-1 (UbxCbx-1), which partially transforms Drosophila wings into halteres via ectopic activation of Ubx. This led us to investigate the potential role of this lncRNA in regulation of Ubx expression. The UbxCbx-1 mutation dramatically changes the pattern of lncRNA: PS4, eliminating the expression of most lncRNA: PS4 sequences from parasegment 4 (where Ubx protein is normally absent) and ectopically activating lncRNA: PS4 at high levels in the abdomen (where Ubx is normally expressed). These changes, however, did not lead to changes in the Ubx embryonic transcription pattern. Targeted deletion of the two promoters of lncRNA: PS4 did not result in the change of Ubx expression in the embryos. In the genetic background of a UbxCbx-1 mutation, the lncRNA: PS4 mutation does slightly enhance the ectopic activation of Ubx protein expression in wing discs and also slightly enhances the wing phenotype seen in UbxCbx-1 heterozygotes.

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings

“…Of note, a systematic in-situ hybridization survey ( Pease et al, 2013 ) showed that Drosophila embryos express an antisense transcript in its 5’ region ( lncRNA:bxd ), as well as within its first intron ( lncRNA:PS4 ). It is thought that Drosophila bxd regulates Ubx , possibly by transcriptional interference or by facilitation of the Fub-1 boundary effect ( Petruk et al, 2006 ; Ibragimov et al, 2023 ), while the possible regulatory roles of PS4 remain debated ( Hermann et al, 2022 ). While these dipteran non-coding transcripts lack detectable sequence similarity with the lepidopteran IT1 and AS5’ transcripts, further comparative genomics analyses of the Ubx region across the holometabolan insect phylogeny should clarify the extent to which Hox cluster lncRNAs have been conserved or independently evolved.…”

“…Fub-1/bxd long non-coding RNAs (lncRNAs) situated 5’ of Ubx are thought to participate in Ubx regulation in the PS5 (posterior T3 to anterior A1) parasegment ( Petruk et al, 2006 ; Ibragimov et al, 2023 ). An intronic lncRNA dubbed lncRNA:PS4 is expressed in the PS4 parasegment (posterior T2 - anterior T3), and appears to stabilize Ubx in this region in mutant contexts ( Hermann et al, 2022 ). Little is known about how insect Hox genes are regulated outside of Drosophila , where they co-localize into a single Hox cluster, and where Antp and Ubx thus occur in contiguous positions ( Gaunt, 2022 ; Mulhair and Holland, 2024 ).…”

Cis-regulatory modes ofUltrabithoraxinactivation in butterfly forewings

“…Of note, a systematic in-situ hybridization survey (Pease et al 2013) showed that Drosophila embryos express an antisense transcript in its 5’ region ( lncRNA:bxd ), as well as within its first intron ( lncRNA:PS4 ). It is thought that Drosophila bxd regulates Ubx , possibly by transcriptional interference or by facilitation of the Fub-1 boundary effect (Petruk et al 2006; Ibragimov et al 2023), while the possible regulatory roles of PS4 remain debated (Hermann et al 2022). While these dipteran non-coding transcripts lack detectable sequence similarity with the lepidopteran IT1 and AS5’ transcripts, further comparative genomics analyses of the Ubx region across the holometabolan insect phylogeny should clarify the extent to which Hox cluster lncRNAs have been conserved or independently evolved.…”

“…Fub-1/bxd long non-coding RNAs (lncRNAs) situated 5’ of Ubx are thought to participate in Ubx regulation in the PS5 (posterior T3 to anterior A1) parasegment (Petruk et al 2006; Ibragimov et al 2023). An intronic lncRNA dubbed lncRNA:PS4 is expressed in the PS4 parasegment (posterior T2 - anterior T3), and appears to stabilize Ubx in this region in mutant contexts (Hermann et al 2022). Little is known about how insect Hox genes are regulated outside of Drosophila , where they co-localize into a single Hox cluster, and where Antp and Ubx thus occur in contiguous positions (Gaunt 2022; Mulhair and Holland 2022).…”

Noncoding RNA Regulation of Hormonal and Metabolic Systems in the Fruit Fly Drosophila

“…Ultrabithorax ( Ubx ), Abdominal-A ( Abd-A ), and Abdominal-B ( Abd-B ) are known for the spatiotemporal expression pattern in development and its mutants will have disrupted abdominal segments [ 127 ]. However, for lncRNA:PS4 , despite having the same expression profile as Ubx , promoter deletion in the lncRNA:PS4 mutant shows no direct expression difference in Ubx [ 140 ]. Another lncRNA, acal , is shown to be responsible for dorsal closure through the JNK signalling pathway, and mutations in acal are lethal [ 128 ].…”