Functional characterization and gene expression profiling of Drosophila melanogaster short dADA2b isoform-containing dSAGA complexes

The data presented here are in accord with results of genetic complementation experiments, and support the hypothesis that different isoforms of dADA2b contribute to the functional variations of dSAGA multiprotein HAT complexes

The dissociable RPB4 subunit of RNA Pol II has vital functions in Drosophila

RNA polymerase II (Pol II) is composed of a ten subunit core and a two subunit dissociable subcomplex comprising the fourth and seventh largest subunits, RPB4 and RPB7. The evolutionary highly conserved RPB4/7 heterodimer is positioned in the Pol II such that it can make contact with various factors involved in RNA biogenesis and is believed to play roles both during the process of transcription and post-transcription. A detailed analysis of RPB4/7 function in a multicellular eukaryote, however, is lacking partly because of the lack of a suitable genetic system. Here, we describe generation and initial analysis of Drosophila Rpb4 mutants. In the fly, RPB4 is a product of a bicistronic gene together with the ATAC histone acetyltransferase complex constituent ADA2a. DmAda2a and DmRpb4 are expressed during fly development at different levels. The structure of mature mRNA forms suggests that the production of DmADA2a and DmRPB4-specific mRNAs is ensured by alternative splicing. Genetic analysis indicates that both DmRPB4 and DmADA2a play essential roles, because their absence results in lethality in early and late larval stages, respectively. Upon stress of high temperature or nutritional starvation, the levels of RPB4 and ADA2a messages change differently. RPB4 colocalizes with Pol II to several sites on polytene chromosomes, however, at selected locus, the abundances of Pol II and RPB4 vary greatly. Our data suggest no tight functional link between DmADA2a and DmRPB4, and reveal differences in the abundances of Pol II core subunits and RPB4 localized at specific regions on polytene chromosomes, supporting the suggested role of RPB4 outside of transcription-engaged Pol II complexes

In vivo effects of abolishing the single canonical sumoylation site in the C-terminal region of Drosophila p53

Using yeast two-hybrid screens we determined that Drosophila (Dm)p53 interacts with proteins involved in sumoylation (UBA2, UBC9 and PIAS) through different regions of its C-terminal domain. A K302R point mutation within a single canonical sumoylation site of Dmp53 did not abolish the observed interactions. These observations prompted us to analyze whether Dmp53 sumoylation at this site has any functional role in vivo. Genetic assays showed that deleting one copy of genes involved in sumoylation (lwr, Su(var)2–10 or smt3 heterozygosity) enhanced slightly the mutator phenotype of Dmp53. We compared the in vivo effects of wild type and K302R Dmp53 overproduced from transgenes and determined that similar levels of expression of the mutant and wild type proteins resulted in similar phenotype, and the two proteins showed similar cellular localization. The half life and the trans-activator activity of K302R mutant and wild type Dmp53 were also comparable. Lastly, by analyzing wild type and K302R Dmp53 expressed at different levels in animals and in S2 cells we detected no differences between the mobility of the mutant and wild-type protein. From these data we conclude that under normal developmental conditions the loss of SUMO modification at K302 does not affect Dmp53 function significantly