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To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

Even in well-characterized genomes, many transcripts are considered noncoding RNAs (ncRNAs) simply due to the absence of large open reading frames (ORFs). However, it is now becoming clear that many small ORFs (smORFs) produce peptides with important biological functions. In the process of characterizing the ribosomebound transcriptome of an important cell type of the seminal fluid-producing accessory gland of Drosophila melanogaster, we detected an RNA, previously thought to be noncoding, called male-specific abdominal (msa). Notably, msa is nested in the HOX gene cluster of the Bithorax complex and is known to contain a micro-RNA within one of its introns. We find that this RNA encodes a "micropeptide" (9 or 20 amino acids, MSAmiP) that is expressed exclusively in the secondary cells of the male accessory gland, where it seems to accumulate in nuclei. Importantly, loss of function of this micropeptide causes defects in sperm competition. In addition to bringing insights into the biology of a rare cell type, this work underlines the importance of small peptides, a class of molecules that is now emerging as important actors in complex biological processes. [ABSTRACT FROM AUTHOR]

DNA-bound transcription factors (TFs) governing developmental gene regulation have been proposed to recruit Polymerase II machinery at gene promoters through specific interactions with dedicated subunits of the evolutionarily-conserved Mediator complex (MED). However, whether such MED subunit specific functions and partnerships have been conserved during evolution has been poorly investigated. To address this issue, we generated the first Drosophila loss-of-function mutants for Med1, known as a specific cofactor for GATA TFs and hormone nuclear receptors in mammals. We show that Med1 is required for cell proliferation, and hematopoietic differentiation depending on the GATA TF Serpent (Srp). Med1 binds physically Srp in cultured cells and in vitro through its conserved GATA Zinc Finger DNA-binding domain and the divergent Med1 C-terminal. Interestingly, GATA/Srp interaction occurs through the longest Med1 isoform, suggesting a functional diversity of MED complex populations. Furthermore, we show that Med1 acts as a coactivator for the GATA factor Pannier during thoracic development. In conclusion, the Med1 requirement for GATA-dependent regulatory processes is a common feature in insects and mammals, although binding interfaces have diverged. Further work in Drosophila should bring valuable insights to fully understand GATA-MED functional partnerships, which probably involve other MED subunits depending on the cellular context. [ABSTRACT FROM AUTHOR]

Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded “co-factor” that acts together with Hox proteins through their homeodomains in regulated developmental transcription. [ABSTRACT FROM AUTHOR]