Regulation of the Drosophila transcriptome by Pumilio and the CCR4-NOT deadenylase complex.

The sequence-specific RNA-binding protein Pumilio controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells in culture. We also use an improved RNA co-immunoprecipitation method to identify Pumilio-bound mRNAs in Drosophila embryos. Integration of these datasets with the locations of Pumilio binding motifs across the transcriptome reveal novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observe concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling reveals that Pumilio binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pumilio-mediated repression are not influenced by their content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase.

The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.

A conserved domain of Drosophila RNA-binding protein Pumilio interacts with multiple CCR4-NOT deadenylase complex subunits to repress target mRNAs.

Pumilio is a sequence-specific RNA-binding protein that controls development, stem cell fate, and neurological functions in Drosophila. Pumilio represses protein expression by destabilizing target mRNAs in a manner dependent on the CCR4-NOT deadenylase complex. Three unique repression domains in the N-terminal region of Pumilio were previously shown to recruit CCR4-NOT, but how they do so was not well understood. In this study, we identified the motifs that are necessary and sufficient for the activity of the third repression domain of Pumilio, designated RD3, which is present in all isoforms and has conserved regulatory function. We identified multiple conserved regions of RD3 that are important for repression activity in cell-based reporter gene assays. Using yeast two-hybrid assays, we show that RD3 contacts specific regions of the Not1, Not2, and Not3 subunits of the CCR4-NOT complex. Our results indicate that RD3 makes multivalent interactions with CCR4-NOT mediated by conserved short linear interaction motifs. Specifically, two phenylalanine residues in RD3 make crucial contacts with Not1 that are essential for its repression activity. Using reporter gene assays, we also identify three new target mRNAs that are repressed by Pumilio and show that RD3 contributes to their regulation. Together, these results provide important insights into the mechanism by which Pumilio recruits CCR4-NOT to regulate the expression of target mRNAs.

Reversible neuronal and muscular toxicity of caffeine in developing vertebrates.

This study utilizes zebrafish embryos to understand the cellular and molecular mechanisms of caffeine toxicity in developing vertebrate embryos. By using a high concentration of caffeine, we observed almost all the phenotypes that have been described in humans and/or in other animal models, including neural tube closure defect, jittery, touch insensitivity, and growth retardation as well as a drastic coiled body phenotype. Zebrafish embryos exposed to 5mM caffeine exhibited high frequent movement, 10 moves/min comparing with around 3 moves/min in control embryos, within half an hour post exposure (HPE). They later showed twitching, uncoordinated movement, and eventually severe body curvature by 6HPE. Exposure at later stages resulted in the same phenotypes but more posteriorly. Surprisingly, when caffeine was removed before 6HPE, the embryos were capable of recovering but still exhibited mild curvature and shorter bodies. Longer exposure caused irreversible body curvature and lethality. These results suggest that caffeine likely targets the neuro-muscular physiology in developing embryos. Immunohistochemistry revealed that the motorneurons in treated embryos developed shorter axons, abnormal branching, and excessive synaptic vesicles. Developing skeletal muscles also appeared smaller and lacked the well-defined boundaries seen in control embryos. Finally, caffeine increases the expression of genes involved in synaptic vesicle migration. In summary, our results provide molecular understanding of caffeine toxicity on developing vertebrate embryos.