Parcas, a regulator of non-receptor tyrosine kinase signaling, acts during anterior-posterior patterning and somatic muscle development in Drosophila melanogaster

We have isolated parcas (pcs) in a screen to identify novel regulators of muscle morphogenesis. Pcs is expressed in the ovary and oocyte during oogenesis and again in the embryo, specifically in the developing mesoderm, throughout muscle development. pcs is first required in the ovary during oogenesis for patterning and segmentation of the early Drosophila embryo due primarily to its role in the regulation of Oskar (Osk) levels. In addition to the general patterning defects observed in embryos lacking maternal contribution of pcs, these embryos show defects in Wingless (Wg) expression, causing losses of Wg-dependent cell types within the affected segment. pcs activity is required again later during embryogenesis in the developing mesoderm for muscle development. Loss and gain of function studies demonstrate that pcs is necessary at distinct times for muscle specification and morphogenesis. Pcs is predicted to be a novel regulator of non-receptor tyrosine kinase (NRTK) signaling. We have identified one target of Pcs regulation, the Drosophila Tec kinase Btk29A. While Btk29A appears to be regulated by Pcs during its early role in patterning and segmentation, it does not appear to be a major target of Pcs regulation during muscle development. We propose that Pcs fulfils its distinct roles during development by the regulation of multiple NRTKs.     

Temporal complexity within a translational control element in the nanos mRNA

Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. We show that both components to spatial control of nanos translation initiate during oogenesis and that translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3' untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. We propose that nanos evolved to exploit two different stage-specific translational regulatory mechanisms.     

p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-regulated genes

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Here we have taken advantage of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), we depleted potential corepressors and looked for the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, our results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. We found that the chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulated the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but had no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins.     

Activation and repression functions of an SR splicing regulator depend on exonic versus intronic-binding position

SR proteins and related factors play widespread roles in alternative pre-mRNA splicing and are known to promote splice site recognition through their Arg-Ser-rich effector domains. However, binding of SR regulators to some targets results in repression of splice sites through a distinct mechanism. Here, we investigate how activated and repressed targets of the Drosophila SR regulator Transformer2 elicit its differing effects on splicing. We find that, like activation, repression affects early steps in the recognition of splice sites and spliceosome assembly. Repositioning of regulatory elements reveals that Tra2 complexes that normally repress splicing from intronic positions activate splicing when located in an exon. Protein tethering experiments demonstrate that this position dependence is an intrinsic property of Tra2 and further show that repression and activation are mediated by separate effector domains of this protein. When other Drosophila SR factors (SF2 and Rbp1) that activate splicing from exonic positions were tethered intronically they failed to either activate or repress splicing. Interestingly, both activities of Tra2 favor the exonic identity of the RNA sequences that encompass its binding sites. This suggests a model in which these two opposite functions act in concert to define both the position and extent of alternatively spliced exons.