Alternative Splicing Modulates Ubx Protein Function inDrosophila melanogaster

Abstract

The Drosophila Hox gene Ultrabithorax (Ubx) produces a family of protein isoforms through alternative splicing. Isoforms differ from one another by the presence of optional segments-encoded by individual exons-that modify the distance between the homeodomain and a cofactor-interaction module termed the ''YPWM'' motif. To investigate the functional implications of Ubx alternative splicing, here we analyze the in vivo effects of the individual Ubx isoforms on the activation of a natural Ubx molecular target, the decapentaplegic (dpp) gene, within the embryonic mesoderm. These experiments show that the Ubx isoforms differ in their abilities to activate dpp in mesodermal tissues during embryogenesis. Furthermore, using a Ubx mutant that reduces the full Ubx protein repertoire to just one single isoform, we obtain specific anomalies affecting the patterning of anterior abdominal muscles, demonstrating that Ubx isoforms are not functionally interchangeable during embryonic mesoderm development. Finally, a series of experiments in vitro reveals that Ubx isoforms also vary in their capacity to bind DNA in presence of the cofactor Extradenticle (Exd ). Altogether, our results indicate that the structural changes produced by alternative splicing have functional implications for Ubx protein function in vivo and in vitro. Since other Hox genes also produce splicing isoforms affecting similar protein domains, we suggest that alternative splicing may represent an underestimated regulatory system modulating Hox gene specificity during fly development.

Integration of RNA processing and expression level control modulates the function of the Drosophila Hox gene Ultrabithorax during adult development

“…Based on our data and on previous reports we think that there may be three different responses when either the Ia or IVa proteins are expressed: (1) some Ubx targets, such as decapentaplegic in the visceral mesoderm (at least as to its anterior repression) and those needed to specify the embryonic cuticle, probably respond similarly to the different Ubx proteins (Busturia et al, 1990;Mann and Hogness, 1990;Subramaniam et al, 1994;Gebelein et al, 2002;Reed et al, 2010); (2) other targets, such as sal, wg and ara in the haltere disc, as well as others needed to repress wing development and promote haltere development, are more efficiently regulated by isoform Ia than IVa, but if the levels of the latter are increased, a similar regulation is achieved; and (3) the expression of dpp in the posterior visceral mesoderm (Reed et al, 2010) or of targets needed to specify the segmental pattern in the embryonic peripheral nervous system (Mann and Hogness, 1990;Subramaniam et al, 1994), are differently controlled by proteins Ia and IVa, independently of their levels of expression.…”

“…Although Dll expression in Ubx MX17 animals, or in embryos with high levels of the IVa isoform, has not been detailed, high levels of this protein repress the formation of Keilin's organs, which is driven by Dll expression (Mann and Hogness, 1990). We note that Ubx MX17 adults have abnormalities in metathoracic legs and the A1 (Busturia et al, 1990; and this report), in the development of A1 and A2 larval abdominal muscles (Reed et al, 2010) and are not as healthy as wild-type flies (Subramaniam et al, 1994), suggesting that isoform IVa may be less efficient than isoform Ia in controlling many Ubx targets, not just those making a haltere. In conclusion, we think that the particular architecture of cis-regulatory regions in each Ubx target may account for the inclusion of each gene in one of these three categories, so a specific research may be needed for each case.…”

The regulation of Hox gene expression during animal development

“…Further genetic work on the PNS showed that indeed Ubx-IVa cannot substitute functionally for other isoforms to promote normal development of the PNS (Subramaniam et al, 1994). Recent studies using the Drosophila UAS/Gal4 system further demonstrated the impact of Ubx alternative splicing on the activation of Ubx target genes during embryogenesis (Reed et al, 2010) and on the ability of Ubx to control the morphology and underlying gene networks of adult appendage development (de Navas et al, 2011).…”

“…Development 140 (19) (Bantignies et al, 2011;Tolhuis et al, 2011) Mouse: (Noordermeer et al, 2011;Yu et al, 1995;Yu et al, 1998) Transcriptional regulation Yes Yes Drosophila: (reviewed by Alexander et al, 2009;Maeda and Karch, 2009;Tschopp and Duboule, 2011) Mouse: (reviewed by Alexander et al, 2009;Maeda and Karch, 2009;Tschopp and Duboule, 2011) lncRNA regulation Yes Yes Drosophila: (Petruk et al, 2006;Sanchez-Elsner et al, 2006) Mouse: Dinger et al, 2008;Wang et al, 2011) RNA processing Yes Yes Drosophila: O'Connor et al, 1988;Hatton et al, 1998;Reed et al, 2010;Thomsen et al, 2010) Mouse: (LaRosa andGudas, 1988;Benson et al, 1995;Fujimoto et al, 1998) miRNA regulation Yes Yes Drosophila: (Ronshaugen et al, 2005;Tyler et al, 2008;Stark et al, 2008;Bender, 2008;Thomsen et al, 2010) Mouse: (Yekta et al, 2004;McGlinn et al, 2009;He et al, 2011) Translational regulation Likely Yes Drosophila: (Oh et al, 1992;Ye et al, 1997) Mouse: (Brend et al, 2003;Kondrashov et al, 2011) Note that owing to space limitations only a few key references are listed here. For further details, see main text.…”

Selection of distinct Hox–Extradenticle interaction modes fine-tunes Hox protein activity

“…These observations indicate that, in the case of Dll regulation, modifying the mode of interaction does not affect DNA binding. However, the distinct DNA binding affinities displayed by Dm-UbxIa and Dm-UbxIVa on a composite Hox/Exd site (34) suggest that modifying the mode of interaction may have a different effect on other targets.…”