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A comprehensive, quantified identification of the mRNA-binding and whole cell proteomes in the three main Leishmanialifecycle stages, the first such comparison in kinetoplastid parasites, demonstrates trans-regulator RBPs select distinct, specific mRNA target pools in a stage-regulated manner despite equivalent, constitutive transcript levels available. Results further indicate that in L. mexicanaparasites, mRNA levels are not a strong predictor of whole cell expression or RNA binding potential of encoded proteins. Included are the first proteomes from the human-infective metacyclic promastigote stage.

Hairless (H) is identified as a Runt-interacting protein that contributes to transcriptional regulation during segmentation in the Drosophilaembryo. H shares a role with Groucho and other corepressors in maintaining Runt-dependent engrailed repression but, unlike these other factors, is also involved in slp1 repression.

Runt is a vital transcriptional regulator in the developmental pathway responsible for segmentation in the Drosophila embryo. Runt activates or represses transcription in a manner that is dependent on both cellular context and the specific downstream target. Here we identify Hairless (H) as a Runt-interacting molecule that functions during segmentation. We find that H is important for maintenance of engrailed (en) repression as was previously demonstrated for Groucho (Gro), Rpd3, and CtBP. H also contributes to the Runt-dependent repression of sloppy-paired-1 (slp1), a role that is not shared with these other corepressors. We further find distinct roles for these different corepressors in the regulation of other Runt targets in the early Drosophila embryo. These findings, coupled with observations on the distinct functional requirements for Runt in regulating these several different targets, indicate that Runt-dependent regulation in the Drosophila blastoderm embryo relies on unique, target-gene-specific molecular interactions.

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators.

An investigation of the in vivo roles of conserved regions of the DrosophilaRunt protein outside of the DNA-binding Runt domain reveals distinct requirements in different regulatory activities. The conserved VWRPY-containing C-terminus required for repression of only a subset of targets is also found to participate in activation of other targets.